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Title:
ASSAYS AND MEDICAL USES
Document Type and Number:
WIPO Patent Application WO/2019/058136
Kind Code:
A1
Abstract:
The present invention relates to methods of identifying agents that inhibit HSE having potential clinical value, in particular for the prevention and/or treatment of a proliferative disorder. The invention also relates to medical uses of HSE inhibitors in the prevention and/or treatment of a proliferative disorder in which an increase in cell division leads to a pathological accumulation of cells, and to corresponding methods of treatment using HSE inhibitors.

Inventors:
SCHOFIELD, Christopher (University of Oxford, Chemistry Research Laboratory12 Mansfield Road, Oxford Oxfordshire OX1 3TA, OX1 3TA, GB)
Application Number:
GB2018/052701
Publication Date:
March 28, 2019
Filing Date:
September 21, 2018
Export Citation:
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Assignee:
OXFORD UNIVERSITY INNOVATION LIMITED (Buxton Court, 3 West Way, Oxford OX2 0JB, OX2 0JB, GB)
International Classes:
G01N33/574
Domestic Patent References:
WO2005071079A12005-08-04
Other References:
CHARLES K. ALLERSTON ET AL: "The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities", NUCLEIC ACIDS RESEARCH, vol. 43, no. 22, 17 November 2015 (2015-11-17), pages 11047 - 11060, XP055528347, ISSN: 0305-1048, DOI: 10.1093/nar/gkv1256
SOOK Y. LEE ET AL: "Cephalosporins inhibit human metallo [beta]-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo", CHEMICAL COMMUNICATIONS, vol. 52, no. 40, 1 January 2016 (2016-01-01), pages 6727 - 6730, XP055528317, ISSN: 1359-7345, DOI: 10.1039/C6CC00529B
PETTINATI ILARIA ET AL: "The Chemical Biology of Human Metallo-[beta]-Lactamase Fold Proteins", TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER, AMSTERDAM, NL, vol. 41, no. 4, 21 January 2016 (2016-01-21), pages 338 - 355, XP029479789, ISSN: 0968-0004, DOI: 10.1016/J.TIBS.2015.12.007
CASSANDRA L. RETZLAFF ET AL: "Metallo-[beta]-lactamase Domain-Containing Protein 1 (MBLAC1) Is a Specific, High-Affinity Target for the Glutamate Transporter Inducer Ceftriaxone", ACS CHEMICAL NEUROSCIENCE, vol. 8, no. 10, 14 August 2017 (2017-08-14), US, pages 2132 - 2138, XP055528226, ISSN: 1948-7193, DOI: 10.1021/acschemneuro.7b00232
Attorney, Agent or Firm:
HGF LIMITED (4th Floor, Merchant Exchange17-19 Whitworth Street West, Manchester M1 5WG, M1 5WG, GB)
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Claims:
CLAIMS

1 . A method of identifying an agent having potential clinical value in the prevention or treatment of a proliferative disorder, the method comprising:

determining the ability of the agent to inhibit HSE activity, wherein the ability to inhibit HSE activity indicates the test compound is an agent having potential clinical value.

2. The method of claim 1 , wherein the inhibition of HSE activity is determined by assessing:

• presence of HSE substrate degradation products;

• cell phenotype; or

• binding of the agent that inhibits HSE activity.

3. A method according to claim 1 or claim 2, wherein the ability to inhibit HSE activity is determined by assessing the presence of HSE substrate degradation products, wherein a reduction in HSE substrate degradation products indicates inhibition of HSE activity.

4. A method according to any of claims 2 to 3, wherein the degradation product is a product generated by cleavage of a sequence set out in SEQ ID NO: 2.

5. A method according to any of claims 2 to 4, wherein the degradation product is cleaved at a residue corresponding to A82 of SEQ ID NO: 2.

6. A method according to any of claims 2 to 5, wherein in the presence of HSE degradation products is assessed by a method selected from the group consisting of: a radiolabel based assay; a fluorescence based assay; a chromatographic assay; and an antibody assay.

7. A method according to claim 2, wherein the inhibition of HSE activity is determined by assessing phenotype cell of a cell to which the agent has been provided.

8. A method according to claim 7, wherein the cell phenotype is determined by assessing at least one of the group consisting of: a cell cycle marker expressed by the cell; core histones expressed by the cell; and altered RNA sequences of an HSE substrate.

9. A method according to claim 8, wherein the cell phenotype is assessed by assaying for expression of a cell cycle marker selected from the group consisting of: Cyclin D1 ; Cyclin E; TGF- 2; CCND1 ; MY016; TGF^RI ; TP63; and PIM1 .

10. A method according to claim 9, wherein an increase in expression of a cell cycle marker selected from the group consisting of: Cyclin D; Cyclin E; TGF-β; CCND1 ; TGF RI ; and PIM1 indicates inhibition of HSE activity.

1 1 . A method according to claim 9 or claim 10, wherein a decrease in expression of a cell cycle marker selected from the group consisting of: MY016; and TP63 indicates inhibition of HSE activity.

12. A method according to claim 8, wherein the cell phenotype is assessed by assaying for expression of core histones expressed by the cell.

13. A method according to claim 12, wherein a reduction in core histone expression indicates inhibition of HSE activity.

14. A method according to claim 8, wherein the cell phenotype is assessed by assaying for polyadenylation of an HSE substrate.

15. A method according to claim 14, wherein an increase in polyadenylation of an HSE substrate indicates inhibition of HSE activity.

16. A method according to any of claims 7 to 15, wherein the cell phenotype is assessed by a method selected from the group consisting of: an antibody assay; a cell typing assay; a cell proliferation assay; and a cell growth assay.

17. A method according to claim 2, wherein the ability to inhibit HSE activity is determined by assessing binding of the agent that inhibits HSE activity.

18. A method according to claim 17, wherein the binding is binding of the agent to HSE.

19. A method according to claim 18, wherein the agent binds to HSE within the active the active site of HSE and inhibits HSE activity.

20. A method according to claim 18, wherein the agent binds to HSE outside of the active the active site of HSE and inhibits HSE activity.

21 . A method according to any of claims 17 to 20, wherein the agent binds to HSE in manner that is competitive with respect to the HSE substrate.

22. A method according to any of claims 17 to 20, wherein the agent binds to HSE in manner that is non-competitive with respect to the HSE substrate.

23. A method according to claim 17, wherein the binding is binding of the agent to an HSE substrate.

24. A method according to claim 23, wherein the HSE substrate comprises a sequence set out in SEQ ID NO: 2.

25. A method according to any of claims 17 to 24, wherein an increase in binding indicates inhibition of HSE activity.

26. A method according to any of claims 17 to 25, wherein the binding is assessed by a method selected from the group consisting of: surface plasmon resonance assay (SPR); nuclear magnetic resonance (NMR); and fluorescence spectroscopy.

27. An inhibitor of HSE activity for use in the prevention and/or treatment of a proliferative disorder.

28. An inhibitor of HSE activity for use according to claim 27, wherein the inhibitor binds to HSE within the active the active site of HSE and inhibits HSE activity.

29. An inhibitor of HSE activity for use according to claim 27 or claim 28, wherein the inhibitor is a specific inhibitor of HSE activity.

30. An inhibitor of HSE activity for use according to any of claims 27 to 29, wherein the proliferative disorder is selected from the group consisting of: cancer, psoriasis, atherosclerosis, rheumatoid arthritis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver, and immunoproliferative disorders.

31 . An inhibitor of HSE activity for use according to claim 30, wherein the proliferative disorder is cancer or psoriasis.

32. An inhibitor of HSE activity for use according to claim 31 , wherein the proliferative disorder is cancer.

33. A method of preventing and/or treating a proliferative disorder in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of an agent that inhibits HSE activity.

34. A method according to claim 33, wherein the proliferative disorder is selected from the group consisting of: cancer, psoriasis, atherosclerosis, rheumatoid arthritis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver, and immunoproliferative disorders.

35. A method according to claim 34, wherein the proliferative disorder is cancer or psoriasis.

36. A method according to claim 35, wherein the proliferative disorder is cancer.

Description:
ASSAYS AND MEDICAL USES

FIELD OF THE INVENTION

The present invention relates to methods of identifying agents having potential clinical value, in particular for the prevention and/or treatment of a proliferative disorder. The invention also relates to medical uses of HSE inhibitors in the prevention and/or treatment of a proliferative disorder, and to corresponding methods of treatment using HSE inhibitors.

BACKGROUND

Unbalanced proliferation of biological cells is responsible for a number of proliferative disorders, in which an increase in cell division, as compared to cell death, leads to a pathological accumulation of cells.

Cell reproduction is controlled by the cell cycle, which has been extensively characterised, and continues to be the subject of much research.

During S-phase of the cell cycle, DNA is replicated and the production of core histone proteins is coordinated with DNA replication. Metazoan mRNAs that encode for the replication dependent (RD) core histones (H2A, H2B, H3, and H4) lack the normal polyA tail formed by 3' end pre-mRNA cleavage and consequent polyadenylation. Instead, they are subjected to endonucleolytic cleavage on the 3' side of an RNA hairpin (stem loop) producing mRNA with a 3 ' -stem loop (SL), which is exported from the nucleus for use in translation. The same endonuclease that is involved in normal protein-coding pre-mRNA cleavage, i.e. cleavage and poyladenylation specificity factor 73 (CPSF73), is proposed to catalyse RD histone pre-mRNA cleavage. Additional factors specific to RD histone pre-mRNA processing, including stem loop binding protein (SLBP) and the U7 small nuclear ribonucleoprotein (U7snRNP) that binds to a histone downstream element (HDE) are proposed to be involved in CPSF73 targeting to RD pre-histone mRNA.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of identifying an agent having potential clinical value in the prevention and/or treatment of a proliferative disorder, the method comprising: determining the ability of the agent to inhibit HSE activity, wherein the ability to inhibit HSE activity indicates the test compound is an agent having potential clinical value. In a second aspect, the invention provides an inhibitor of HSE activity for use in the prevention and/or treatment of a proliferative disorder.

In a third aspect the invention provides a method of preventing and/or treating a proliferative disorder in a subject in need thereof, the method comprising providing the subject with a therapeutically effective amount of an agent that inhibits HSE activity.

In a fourth aspect, the invention provides a pharmaceutical composition comprising an inhibitor of HSE activity. The inhibitor of HSE activity may be a specific inhibitor of HSE activity.

DESCRIPTION OF FIGURES

Figure 1. HSE is a metallo β-lactamase fold endonuclease.

Figure 2. Loss of HSE leads to 3' end processing defects in RD histone pre-mRNA. Figure 3. Loss of HSE impairs cell cycle progression.

Figure 4. Loss of HSE impairs normal entering to S-phase and its progression.

Figure 5 (Extended Data Figure 1 ) spectrometry deconvoluted spectra of recombinant HSE produced in E. coli

Figure 6 (Extended Data Figure 2). Structural comparison of the HSE with ribonucleases from the MBL superfamily and with glyoxalase II.

Figure 7 (Extended Data Figure 3). HSE localizes to the nuclear envelope and interacts with CLP1.

Figure 8 (Extended Data Figure 4). Loss of HSE impairs normal entering and S-phase progression.

Figure 9 (Extended Data Figure 5). HSE is RD histone pre-mRNA processing endoribonuclease specifically involved in cell cycle regulation

Figure 10 (Extended Data Figure 6). HSE CRISPR/Cas9 stable knockdown validation. Figure 1 1 (Extended Data Figure 7). The 3' end processing defect in RD histone pre-mRNA due to the HSE depletion increases the polyadenylated fraction of histones mRNA and leads to a transcription termination defect in HeLa cells

Figure 12 is a Western blot showing the impact of an HSE inhibitor (designated 382 and 474) on expression of histone H3 in HeLa cells. This demonstrates the ability of an agent identified by a method in accordance with the first aspect of the invention to inhibit intracellular HSE.

Figure 13 demonstrates that HSE shows endoribonucleolytic activity in vitro. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the inventors' new finding that HSE is an endoribonuclease, the action of which is necessary for cell cycle progression. Accordingly, HSE activity is required for division, and proliferation, of biological cells.

Once this function of HSE is known, it becomes possible to use the ability to inhibit HSE as a screen to identify agents that may have therapeutic utility, for instance in the prevention and/or treatment of proliferative disorders. The skilled person will appreciate that inhibition of HSE activity may be demonstrated by any suitable indicator. In particular, inhibition of HSE activity of the sort that indicates potential therapeutic utility may be demonstrated by the ability to bring about a reduction in the ability of HSE to function as endoribonuclease.

Merely by way of example, the inventors have shown that inhibition of HSE activity may readily be determined by exposing a test system comprising HSE and one or more components capable of interacting with HSE to a test compound, and then assessing one or more of the following indicators:

• the presence of HSE substrate degradation products;

• cell phenotype; or

• binding of the agent that inhibits HSE activity.

These non-limiting examples are considered in further detail elsewhere in the present specification. Once an agent has been identified as being an HSE inhibitor, it is then known to possess at least one property that indicates that it may have therapeutic utility, for example in the prevention and/or treatment of proliferative disorders, such as cancer or psoriasis. Further details of the biological activities and/or physical and chemical properties of the agent in question can then be investigated in order to further determine the likely usefulness of the potential drug.

It will be appreciated that, once the ability to inhibit HSE activity, and thereby inhibit cell cycle progression, has been established, an agent may be used in medical uses or methods of treatment, such as those set out in the second and third aspects of the invention. These aspects relate to the use of such agents in the prevention and/or treatment of proliferative disorders. Examples of such proliferative disorders to be prevented or treated include cancer and psoriasis, though further exemplary conditions are considered elsewhere in the specification.

The invention will now be described further. To aid the understanding of the invention, certain terms used in the present disclosure will be defined in the following paragraphs.

HSE

Histone specific endonuclease (HSE) is a member of the metallo β lactamase fold (MBL-fold) protein family, also known as MBL domain containing protein 1 (MBLAC1 ). The amino acid sequence of human HSE is set out in SEQ ID NO.1 .

The inventors have found that HSE activity is necessary for progression through the S-phase of the cell cycle and subsequently cell division and cell proliferation.

As first demonstrated by the inventors, HSE is an endoribonuclease that catalyses RNA degradation. Additionally, HSE has a di-zinc ion containing active site and catalyses replication dependant (RD) histone pre-RNA cleavage and is specific for RD histone pre-mRNA cleavage. The inventors have found that HSE activity is required for the processing of RD histone pre- RNA. Depletion of HSE from cells leads to the production of unprocessed RD histone pre- mRNA due to inefficient 3' end processing. Additionally, the presence of core histones correlates with cell cycle progression.

Since HSE activity is necessary for production of core histone proteins and cell cycle progression, it is also essential in cell division and cell proliferation. Therefore, HSE activity has particular utility as a target protein in the screening of agents that have the ability inhibit cell proliferation and have therapeutic utility in the prevention and treatment of proliferative disorders.

HSE ACTIVITY

The present invention makes use of HSE and HSE activity as a target in the screening of agents having therapeutic utility in the prevention and treatment of proliferative disorders, as well as using such agents to therapeutically impair HSE activity.

Without wishing to be bound by any hypothesis, the inventors believe that it HSE's activity as an endoribonuclease that confers its role in the control of cell proliferation. Accordingly, HSE inhibition in the context of the present disclosure may refer to any detectable decrease in HSE's ability to function as an endoribonuclease.

This function may be demonstrated by HSE's ability to degrade a substrate comprising the restriction site cleaved by HSE (such a molecule is also referred to herein as "an HSE substrate", and discussed further below). Accordingly, it will be recognised that HSE activity may result in the formation of degradation products of the HSE substrate. Additionally, HSE activity may be determined by assessing cell phenotype, including markers of cell cycle progression and binding of HSE to an HSE substrate.

As previously discussed, HSE activity is necessary for cell cycle progression and therefore cell division and cell proliferation. There are a number of pathological conditions (herein referred to as proliferative disorders) that arise as a result of cell proliferation. An agent that has the ability to inhibit HSE activity, and thus cell proliferation, will therefore have potential clinical value, for example in the prevention and/or treatment of proliferative disorders.

The endoribonuclease activity of HSE may be considered the "direct" activity of this enzyme associated with cell cycle progression. This then leads, in turn, to further "indirect" activities of HSE, which include regulation of polyadenylation, changes in expression of cell cycle markers, and expression of core histones.

Since both direct and indirect activities of HSE each require binding of the enzyme to its substrate, it will also be appreciated that agents that reduce such binding are also able to inhibit HSE activity. Inhibition of HSE activity may be demonstrated with reference to any one or more of the activities of HSE considered above, and with reference to any combination of such activities. Inhibition of HSE activity is discussed further below.

INHIBITION OF HSE ACTIVITY

The inventors have found that inhibition of HSE activity is able to reduce cell proliferation. As discussed elsewhere in this specification there are many means by which a skilled person may determine the ability of an agent to inhibit HSE activity, for example in a method of the first aspect of the invention. These methods may also be used to demonstrate or investigate the extent of inhibition that may be achieved using a known HSE inhibitor (for example to be employed in the medical uses or methods of treatment of the second or third aspects of the invention).

Inhibition of HSE activity may be determined by assessing one or more of the following indicators: presence of HSE substrate degradation products; cell phenotype; or binding of the agent that inhibits HSE activity.

Where desired, inhibition of HSE activity may be quantitatively or qualitatively determined by assessing HSE activity compared to an obtained reference value. An obtained value lower than the reference value may be indicative that HSE activity is inhibited. Inhibition of HSE activity may result in a reduction of 5% or more in HSE activity when compared to a reference value. Inhibition of HSE activity results in a reduction of 10% or more, a reduction of 15% or more, a reduction of 20% or more, a reduction of 25% or more, a reduction of 30% or more, a reduction of 35% or more, a reduction of 40% or more, a reduction of 45% or more, a reduction of 50% or more, a reduction of 55% or more, a reduction of 60% or more, a reduction of 70% or more, a reduction of 75% or more, a reduction of 80% or more, a reduction of 85% or more, a reduction of 90% or more, a reduction of 95% or more, or a reduction of up to 100% in HSE activity when compared to a reference value. Such reductions provide an indication that the test compound is an agent having potential clinical value.

Inhibition of HSE activity may result in at least a 5% reduction in HSE activity when compared to a reference value. Inhibition of HSE activity results in at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction or at least a 100% reduction in HSE activity when compared to a reference value.

HSE SUBSTRATES AND DEGRADATION PRODUCTS

HSE is an endoribonuclease capable of catalysing degradation of substrates that comprise its corresponding restriction site. For brevity, such substrates are referred to as "HSE substrate" herein.

An example of an HSE substrate is set out in SEQ ID NO: 2. This is a 168 nucleotide RNA fragment derived from human histone gene 2H3C (HIST2H3C) with a 5' T7 RNA polymerase promotor sequence. The HSE substrate of SEQ ID NO: 2 comprises the HSE restriction site, and the catalytic activity of HSE is able to cleave SEQ ID NO: 2 at base A82.

Suitably, the degradation product is a product generated by cleavage of a sequence set out in SEQ ID NO: 2. The degradation product may comprise a sequence set out in SEQ ID NO: 2.

Suitably, the degradation product is cleaved at a residue corresponding to A82 of SEQ ID NO: 2. Degradation products may comprise a sequence ending with a base corresponding to A82 of SEQ ID NO:2. Additionally or alternatively, degradation products may comprise a sequence beginning with a base corresponding to C83 of SEQ ID NO:2.

The inventors believe that, after initially cleaving its substrate at a base corresponding to A82 of SEQ ID NO:2, HSE may then continue to hydrolyse the resulting degradation products, digesting these further.

Suitable techniques by which the presence of a degradation product may be recognised are described elsewhere in the specification. These may include assessment by a method selected from the group consisting of: a radiolabel based assay; a fluorescence based assay; a chromatographic assay; and an antibody assay.

Purely by way of example, suitable techniques may include generation of fragments having the predicted molecular weight of a degradation product, or loss of an epitope that occurs on cleavage of the restriction site (for example an epitope spanning A82 and C83). Alternatively, and as discussed further in the Examples, suitable techniques may include incubation of radiolabeled RNA with HSE, fractionation of the resulting degradation products using polyacrylamide gel. The polyacrylamide gel may then be analysed visually and by densitometry.

Inhibition of HSE activity may be demonstrated by a decrease in the presence or quantity of HSE substrate degradation products produced in the presence of an agent having HSE inhibitory activity, as compared to the presence or quantity of such degradation products produced under comparison conditions. Suitably the comparison conditions may be control conditions in which an HSE inhibitor is absent.

Inhibition of HSE activity may be determined by assessing quantifying the presence of HSE substrate degradation products present, and comparing the value obtained to an obtained reference value.

An obtained value lower than the reference value may be indicative that HSE activity is inhibited. Inhibition of HSE activity may result in at least a 5% reduction in degradation products when compared to a reference value. Inhibition of HSE activity results in at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction or at least a 100% reduction in degradation products when compared to a reference value and indicates the test compound is an agent having potential clinical value.

CELL PHENOTYPE

In a suitable embodiment, inhibition of HSE activity is determined by assessing the cell phenotype of a cell to which an agent has been provided. Suitably the cell phenotype is determined by assessing at least one parameter selected from the group consisting of:

• a cell cycle marker expressed by the cell;

• core histones expressed by the cell; and

• altered RNA sequences of an HSE substrate. Suitably a combination of two or even three of these parameters may be assessed in a method of the invention. Inhibition of HSE activity may be demonstrated in respect of one, two, or three of the parameters assessed.

Further details of the manner in which these cell phenotypes may be assessed, and the presence or absence of inhibition of HSE activity determined, are set out in the following paragraphs.

Cell cycle markers

In a suitable embodiment, the cell phenotype is assessed by assaying for expression of a cell cycle marker selected from the group consisting of: Cyclin D1 ; Cyclin E; Transforming growth factor beta 2 gene (TGF-32); cyclin D1 gene (CCND1 ); myosin 16 gene (MY016); Transforming growth factor beta receptor 1 gene (TGF- R1 ); tumour protein 63 gene (TP63); and Proto-oncogene serine/threonine-protein kinase gene (PIM1 ). Expression of one, two, three, four, five, six, seven, or all eight of these markers may be used in the methods of the invention.

These markers are well known to those skilled in the art, and suitable techniques by which their expression may be determined, and quantified if desired, will be known to those wishing to practice the invention.

Expression of certain of these cell cycle markers increases when HSE activity is inhibited. For example, an increase in expression of a cell cycle marker selected from the group consisting of: Cyclin D; Cyclin E; TGF-β; CCND1 ; ΤΰΡ ΡΙΙ ; and PIM1 indicates inhibition of HSE activity. Suitably an increase in expression may be observed in respect of at least one of the markers assessed. An increase in expression may be observed in respect of more than one of the markers assessed. Indeed, an increase in expression may be observed in respect of each of the markers assessed.

By way of example, a one-fold increase in expression may be observed in respect of at least one of the markers assessed. Suitably a one-fold increase in expression may be observed in respect of more than one of the markers assessed. Indeed, a one-fold increase in expression may be observed in respect of each of the markers assessed.

In a suitable embodiment, a two-fold increase in expression may be observed in respect of at least one of the markers assessed. A two-fold increase in expression may be observed in respect of more than one of the markers assessed. Suitably, a two-fold increase in expression may be observed in respect of each of the markers assessed.

Expression of other cell cycle markers decreases when HSE activity is inhibited. For example, decrease in expression of a cell cycle marker selected from the group consisting of: MY016; and TP63 indicates inhibition of HSE activity. Suitably a decrease in expression may be observed in respect of at least one of the markers assessed. Suitably a decrease in expression may be observed in respect of both of the markers assessed.

A one-fold decrease in expression may be observed in respect of at least one of the markers assessed. Suitably, a one-fold decrease in expression may be observed in respect of more than one of the markers assessed. Indeed, a one-fold decrease in expression may be observed in respect of each of the markers assessed.

A two-fold decrease in expression may be observed in respect of at least one of the markers assessed. A two-fold decrease in expression may be observed in respect of more than one of the markers assessed. In certain embodiments, a two-fold decrease in expression may be observed in respect of each of the markers assessed.

A three-fold decrease in expression may be observed in respect of at least one of the markers assessed. Indeed, three-fold decrease in expression may be observed in respect of more than one of the markers assessed. In a suitable embodiment, a three-fold decrease in expression may be observed in respect of each of the markers assessed.

Core histones

Cells contain four "core histone" proteins: H2A; H2B; H3; and H4. As referred to above, the inventors have identified that HSE is specific for replication dependent (RD) histone mRNA cleavage and efficiency of processing at the 3' end. Depletion of HSE in cells results in a reduction in the expression of core histones. HSE depletion also increases polyadenylation of histone mRNA. Accordingly, in a suitable embodiment, phenotype of a cell provided with an agent in a method of the invention may be assessed by assaying for expression of core histones expressed by the cell, or assaying for polyadenylation of histone mRNA.

Suitably, the inhibition of HSE activity may be assessed by assaying for expression of core histones, or changes in mRNA adenylation, after exposure to an agent in a method of the invention. It will be appreciated that expression of one, two, three, or all four of the core histones may be assayed.

In such embodiments a reduction in core histone expression indicates inhibition of HSE activity. Thus an agent that brings about a reduction in core histone expression may be considered an inhibitor of HSE activity (and so an agent with potential therapeutic utility). A reduction in expression may be noted in respect of at least one of the core histones expression of which is being assessed. Suitably a reduction in expression may be noted in respect of more than one, or even each, of the core histones expression of which is assessed.

Alteration of RNA sequences of HSE substrates

The inventors have identified that HSE functions in vivo to alter the RNA sequences of its substrates. In particular, HSE cleaves its substrates at the restriction site.

Inhibition of HSE activity prevents such cleavage, and so may give rise to the accumulation of substrates comprising with a 3' extended sequence. The extension may occur as a result of the presence of polyadenylation of the substrate.

In a suitable embodiment of the methods of the invention, the cell phenotype is assessed by assaying for altered RNA sequence of an HSE substrate. The altered RNA sequence may be the presence of a 3' extended sequence, which is indicative of HSE activity being inhibited. Suitably, the cell phenotype is assessed by assaying for polyadenylation of an HSE substrate.

Since the action of HSE reduces accumulation of polyadenylated substrates in vivo, an increase in polyadenylation of an HSE substrate indicates inhibition of HSE activity. Thus an agent that brings about an increase in polyadenylation of an HSE substrate may be considered an inhibitor of HSE activity (and so an agent with potential therapeutic utility).

ASSAYS

Various assays that may be used in practicing the methods of the invention are discussed elsewhere in the specification. Having been informed of the role of HSE activity in progression through the cell cycle, and the impact of HSE inhibition on cells receiving such treatment, the skilled person will be readily able to determine a suitable assay with reference to the parameter to be assessed. In the case that HSE activity is determined by assessing the presence of HSE substrate degradation products, a suitable assay method may be selected from the group consisting of: a radiolabel based assay; a fluorescence based assay; a chromatographic assay (for example, HPLC); and an antibody assay (for example ELISA, Western blot analysis)

In an embodiment in which cell phenotype is to be assessed by assaying of a cell cycle marker, a suitable assay may be selected from the group consisting of: and antibody assay (for example, ELISA, Western blot, Immunohistochemistry, Immunocytochemistry, Flow cytometry); cell typing assays (for example, Immunoprecipitation, Flow cytometry); cell proliferation assays; cell growth assays.

In the case that HSE activity is to be determined by assessing expression of core histones, expression of such histone proteins may be assessed by radiolabel based assays; or gene analysis (for example RT-qPCR).

In the case that HSE activity is to be determined by assessing binding of an agent that inhibits HSE a suitable assay may be selected from the group consisting of: Surface plasmon resonance assay, nuclear magnetic resonance (NMR) and fluorescence spectroscopy.

Generally, fluorescence based assays, or chromatographic assays (such mass spectrometry) are suitable for investigation of RNA degradation products (for example in in vitro assays), while antibody assays are suitable for investigation of the phenotype of cells after (in vivo) inhibition of HSE (for example with reference to histone and cyclin protein levels).

For the purposes of the present disclosure, antibody-based assays may be taken as encompassing a range of techniques known to those skilled in the art. These include, but are not limited to: immunofluorescence, which may be utilised in flow cytometry assays, such as fluorescence-activated cell sorting; ELISAs (enzyme-linked immunosorbent assays), including variants such as sandwich ELISAs; and blotting-based techniques, such as Western blotting.

Binding

As noted above, the activities of HSE require binding of the enzyme to its substrate, and so agents that reduce such binding are also able to inhibit HSE activity.

In a suitable embodiment, the ability to inhibit HSE activity is determined by assessing binding of the agent. Suitably, an increase in binding indicates inhibition of HSE activity. The skilled person will appreciate that there are a number of different ways in which binding of an agent may inhibit HSE activity, and that accordingly there are a number of different ways in which such binding (and hence inhibition) may be determined. Illustrative examples of these are set out below.

Purely by way of example, binding may be assessed by a method selected from the group consisting of: surface plasmon resonance assay (SPR); nuclear magnetic resonance (NMR); and fluorescence spectroscopy.

Binding to HSE

In a suitable embodiment the binding is binding of the agent to HSE. In such an embodiment the agent may bind to HSE within the active the active site of HSE and thereby inhibit HSE activity. As discussed further (in the Examples and elsewhere in this specification), the inventors have identified a small molecule inhibitor of HSE activity that is able to bind at the active site of HSE. Interestingly, although this inhibitor appears to bind zinc ions present in HSE's active site, the agent is not a zinc chelator. Accordingly, suitable binding may occur via metal ions, such as zinc ions, at the active site of HSE, without metal chelation. By the same token, suitable agents, and HSE inhibitors suitable for use in the methods, medical uses, and methods of treatment of the invention, will include those that bind to the active site of HSE via metal ions, such as zinc ions. Suitably such agents or inhibitors are not zinc chelating agents.

Alternatively, the agent may bind to HSE outside of the active site of HSE and inhibits HSE activity in this manner.

Various forms of binding are known with respect to whether or not the binding of one entity (for example, an agent) is competitive with respect to the binding of another agent (for example, an HSE substrate). In a suitable embodiment the agent binds to HSE in manner that is competitive with respect to binding of the HSE substrate. Alternatively, the agent may bind to HSE in manner that is non-competitive with respect to binding of the HSE substrate.

Binding to an HSE substrate

Alternatively, or additionally, to binding of the agent to HSE itself, HSE activity may be inhibited by an agent that is able to bind HSE's substrate, and thereby reduce activity of the enzyme. Thus, in a suitable embodiment of the methods of the invention, the binding is binding of the agent to an HSE substrate.

Suitably the HSE substrate comprises a sequence set out in SEQ ID NO: 2. Agents and HSE inhibitors

In the present disclosure, "agents" may be referred to in the context of both testing for HSE inhibitory activity (and thus potential clinical value), and also in the context of such medical uses once inhibitory activity has been identified. The medical uses and methods of treatment of the invention also refer to "HSE inhibitors", which may be "agents" in respect of which HSE inhibitory activity has been confirmed.

Any suitable molecule may be used as an agent for the purposes of the present invention. Merely by way of example, a suitable agent may be selected from the group consisting of: a protein agent; a nucleic acid agent; and a small molecule agent.

A suitable protein agent may be a naturally occurring or artificial protein. A suitable protein agent may be an antibody, an antigen-binding fragment of an antibody, or a derivative or variant of such an antibody or fragment thereof.

A nucleic acid agent in the context of the invention may be an antisense oligonucleotide, an siRNA or other interfering or inhibitory nucleic acid; or an aptamer.

An agent or inhibitor may be capable of specifically inhibiting HSE activity. Such a specific inhibitor may be capable of preferentially inhibiting HSE activity, but not the enzyme activity of other MBL fold proteins, or other enzymes.

The skilled reader will be aware of ways in which large molecule inhibitors (such as blocking antibodies, or nucleic acid inhibitors) may be designed to achieve specificity. Selectivity may also be confirmed through screening of agents (whether large or small molecules) in methods analogous to those disclosed herein in respect of HSE, in order to identify those agents that do not undesirable non-specific inhibition.

A suitable small molecule agent may be an inhibitor of HSE, such as the exemplary inhibitor designated 474 referred to in the Examples, that is able to inhibit intracellular HSE. Such an inhibitor may be a specific inhibitor of HSE. The inhibitor may bind to the active site of HSE, thus inhibiting its enzymatic activity.

As referred to elsewhere in the specification, a suitable agent, or suitable HSE inhibitor for use in the medical uses or methods of treatment of the invention, may bind at the active site of HSE via metal ions, particularly via zinc ions. Suitably, such an agent or inhibitor is not a metal chelator (which may be advantageous for a number of reasons, including the risk of undesirably broad non-specific activity of agents that chelate metal ions).

POTENTIAL CLINICAL VALUE

The methods of the invention allow the identification of agents having potential clinical value. This clinical value may be in the prevention and/or treatment of proliferative disorders, as set out elsewhere in the specification.

The link between HSE inhibition and clinical value arises from the inventors' finding that inhibition of HSE activity prevents cell cycle progression.

A proliferative disorder can be any pathological condition that arises from cell proliferation. Cell proliferation is a balance in the body between the production of new cells, by cell division, and cell loss. Cell proliferation can occur as a result of an increase in cell division, and/or a decrease in cell loss. Since inhibition of HSE activity inhibits cell proliferation, so agents that are able to inhibit HSE activity show potential clinical value in the prevention and/or treatment of proliferative disorders.

Proliferative disorders

Cell proliferation results in growth, multiplication or rapid production of tissue or cells and can result pathological conditions. Alternatively or additionally cell proliferation may result in the impairment or loss of tissue function, which for the purposes of the present disclosure may be referred to as proliferative disorders. It can be appreciated that prevention of cell proliferation is beneficial in the treatment of proliferative disorders.

Proliferative disorders can be any pathological condition that arises from cell proliferation. A proliferative disorder can occur in any tissues type; including epithelial tissue, muscular tissue, connective tissue, or nervous tissue. Suitably the proliferative disorder is selected from the group consisting of: cancer, psoriasis, atherosclerosis, rheumatoid arthritis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver, and immunoproliferative disorders.

A proliferative disorder may be a cancer. The cancer may be a solid cancer such as a sarcoma or a carcinoma. Example of solid cancers may include; lung cancer, skin cancer, bowel cancer, breast cancer, prostate cancer, bowel cancer, bladder cancer. Alternatively, the cancer may be a blood cancer, such as leukaemia, myeloma or lymphoma.

The skilled person will also be aware of many deleterious proliferative disorders other than cancer, including, but not limited to, the following examples.

A proliferative disorder may arise from proliferation of cells in the arterial tissue, for example atherosclerosis. A proliferative disorder may result in cell proliferation in the joints, such as rheumatoid arthritis. A proliferative disorder may arise from cell proliferation in the skin, such as psoriasis. A proliferative disorder may arise from cell proliferation in the lungs, for example Idiopathic pulmonary fibrosis. A proliferative disorder may arise from cell proliferation in the connective tissue, for example scleroderma. A proliferative disorder may arise from cell proliferation in the liver cells, for example cirrhosis of the liver. A proliferative disorder may arise from cell proliferation in the immune system causing immunoproliferative disorders, for example myeloproliferative or lymphoproliferative.

Medical uses and methods of treatment of the invention

The second aspect of the invention provides medical uses of an inhibitor of HSE activity, while the third aspect provides methods of treatment employing such inhibitors. Both the medical uses and methods of treatment are of use in the prevention and/or treatment of proliferative disorders.

Suitably the inhibitor of HSE activity for use according to the second aspect of the invention is for use in the prevention and/or treatment of a proliferative disorder by inhibiting HSE activity.

Examples of proliferative disorders that may be prevented and/or treated in accordance with the second or third aspects of the invention are considered above.

In the context of the present invention, "prevention" should be taken as therapeutic intervention that avoids or delays the onset or development of a disorder. In contrast, "treatment" should be taken as therapeutic intervention that alleviates or prevents the progression of an existing disorder.

A subject that may benefit from prevention and/or treatment through the medical uses or methods of treatment of the invention may be identified by an appropriate clinician, with reference to factors such as symptoms, or personal or familial medical history.

Suitable examples of inhibitors of HSE activity, or properties that may be demonstrated by a suitable inhibitor of HSE activity, are described elsewhere in the specification, either in connection with "agents" or "inhibitors".

In a suitable embodiment, an inhibitor of HSE activity for use in the medical uses or methods of treatment of the invention binds to HSE within the active the active site of HSE and inhibits HSE activity.

In a suitable embodiment, an inhibitor of HSE activity for use in the medical uses, or methods of treatment, of the invention is a specific inhibitor of HSE activity.

The small molecule HSE inhibitor identified herein as 474 is an example of an inhibitor suitable for use in the medical uses or methods of treatment of the invention.

Suitable formulations, routes of administration, and doses of HSE inhibitors that may be utilised in the medical uses or methods of treatment of the invention are considered further below.

Pharmaceutical compositions comprising HSE inhibitors

The fourth aspect of the invention provides a pharmaceutical composition comprising an inhibitor of HSE activity. The inhibitor of HSE activity may be a specific inhibitor of HSE activity.

It will be appreciated that pharmaceutical compositions in accordance with the fourth aspect of the invention represent suitable means by which an HSE inhibitor may be utilised in the medical uses of the second aspect of the invention, or provided for the methods of treatment of the fourth aspect of the invention. Formulation of HSE inhibitors for use in the medical uses or methods of treatment of the invention

Also provided by the present invention is a pharmaceutical composition comprising an HSE inhibitor. In embodiments, the composition is a composition comprising the HSE inhibitor and a pharmaceutically acceptable diluent, carrier or excipient. Such compositions may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The compositions may also include antioxidants and/or preservatives. As antioxidants may be mentioned thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid. Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.

The HSE inhibitor may be presented as solids in finely divided solid form, for example they may be micronised. Powders or finely divided solids may be encapsulated.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio..

The HSE inhibitor may be for administration to the subject by any suitable route by which a therapeutically effective amount of HSE inhibitor may be provided.

In one embodiment, the HSE inhibitor is for oral administration. Suitable oral administration forms that may be used in such embodiments include solid dosage forms. Solid dosage forms for oral administration include capsules, tablets (also called pills), powders and granules. In such solid dosage forms, the HSE inhibitor is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules and tablets, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.

Suitably, oral formulations may contain a dissolution aid. The dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkylamine oxides; bile acid and salts thereof (e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface agents, such as sodium laurylsulfate, fatty acid soaps, alkylsulfonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface agents, such as betaines and aminocarboxylic acid salts. Pharmaceutical compositions of the invention, comprising the HSE inhibitor, may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

In one embodiment, the HSE inhibitor is for administration in liquid dosage form. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to HSE inhibitor, the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents. Suspensions, in addition to the HSE inhibitor may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.

In one embodiment the HSE inhibitor may be for administration to the subject by intravenous route. In such an embodiment, a sterile pharmaceutical composition may be especially desirable.

A sterile pharmaceutical composition may be created, for example, by filtration through sterile filtration membranes, prior to or following lyophilisation and reconstitution of the HSE inhibitor. The HSE inhibitor may be stored in lyophilised form or in solution.

A pharmaceutical composition comprising the HSE inhibitor may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierce-able by a hypodermic injection needle.

A sterile pharmaceutical composition comprising the HSE inhibitor suitable for intravenous delivery may be formulated according to conventional pharmaceutical practice as described in Remington: The Science and Practice of Pharmacy (20 th ed, Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.

In a suitable embodiment the pharmaceutical composition comprising the p HSE inhibitor may be for the sustained release of the protein. Such a pharmaceutical composition may comprise semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

In another embodiment, pharmaceutical compositions for sustained release of the HSE inhibitor, may comprise crystals of the HSE inhibitor suspended in suitable formulations capable of maintaining crystals in suspension. Such pharmaceutical compositions, when injected intravenously, subcutaneously or intraperitoneally may produce a sustained release effect.

Routes of administration and dosing using HSE inhibitors

An HSE inhibitor for use in accordance with the medical uses or methods of treatment of the invention may be provided by any suitable route of administration. Examples of such routes of administration have been considered in the preceding paragraphs, in the context of pharmaceutical compositions, and these routes are also to be considered to be disclosed in connection with the medical uses and methods of treatment of the second and third aspects of the invention. Without limitation, the skilled person will be able to select a suitable route of administration with reference to the nature of the proliferative disorder that is to be prevented and/or treated, the subject receiving such prevention and/or treatment, and the nature of the HSE inhibitor to be employed.

The medical uses and methods of treatment of the invention may both make use of a therapeutically effective amount of an HSE inhibitor that is to be provided to a subject requiring prevention and/or treatment of a proliferative disorder. The therapeutically effective amount may be provided via a single incidence of administration, or as a result of multiple incidences of administration.

A requisite therapeutically effective amount of an HSE inhibitor for use in the second or third aspects of the invention may be determined with reference to factors including: reference to the nature of the proliferative disorder that is to be prevented and/or treated; the severity of the disorder; the age or weight of the subject receiving such prevention and/or treatment; and the nature of the HSE inhibitor to be employed.

Merely by way of example, a suitable dose of an HSE inhibitor to be provided in a single incidence of administration to bring about prevention and/or treatment of a proliferative disorder may be between approximately 1 ng and 1 g, A suitable dose may be between approximately 100ng and 500mg. Suitably a dose may be between about 500ng, or even 500pg and 1 mg.

EXAMPLES

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease

During S-phase of the cell cycle, production of core histone proteins is coordinated with DNA replication. Metazoan mRNAs that encode for the replication dependent (RD) core histones (H2A, H2B, H3, and H4) lack the normal polyA tail formed by 3" end pre-m RNA cleavage and consequent polyadenylation. Instead, they are subjected to endonucleolytic cleavage on the 3' side of an RNA hairpin (stem loop) producing mRNA with a 3 ' -stem loop (SL), which is exported from the nucleus for use in translation. The same endonuclease that is involved in normal protein-coding pre-mRNA cleavage, i.e. cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to catalyse RD histone pre-mRNA cleavage. Additional factors specific to RD histone pre-mRNA processing, including stem loop binding protein (SLBP) and the U7 small nuclear ribonucleoprotein (U7snRNP) that binds to a histone downstream element (HDE) are proposed to be involved in CPSF73 targeting to RD pre- histone mRNA. We report that a different histone specific endonuclease (HSE), which like CPSF73 is a metallo- -lactamase (MBL) fold protein, is specific for RD histone pre-mRNA cleavage. Crystallographic and biochemical studies reveal HSE has a di-zinc ion containing active site related to that of CPSF73, but has a distinct overall fold. Notably HSE depletion from cells leads to the production of unprocessed RD histone pre-mRNA due to inefficient 3' end processing. The consequent depletion of core histone proteins correlates with a cell cycle defect due to a delay in entering/progressing through S-phase. HSE thus represents a new type of S-phase specific cancer target.

Cancer medicines, including histone deacetylase and cyclin dependent kinase inhibitors, target the S-phase of the cell cycle. In work aimed at identifying potential new S-phase cancer targets, we considered roles of MBL-fold proteins involved in nucleic acid hydrolysis. In addition, to the role of CPSF73, and the likely pseudo-enzyme CPSF100, in pre-mRNA processing, MBL-fold nucleases are involved in DNA repair (SNM1 A-C) and snRNA and tRNA processing (INTS9 and INTS1 1 , and ELAC 1 and 2, respectively). Whilst most of the -18 human MBL-fold proteins have established functions, several are not assigned, including MBL domain containing protein 1 (MBLAC1 ), renamed here as histone specific endoribonuclease (HSE). On the basis of sequence similarity HSE has been assigned as a glyoxalase II subfamily enzyme (Extended Data Fig. 1 a). However, we found that recombinant HSE prepared from E. coli has only low, likely non-specific, glyoxalase activity as observed for other hMBL-fold proteins belonging to the same subfamily. To investigate HSE function, we solved its crystal structure (1.8 A resolution, P1 space group). Four HSE molecules (chains A-D) are present in the asymmetric unit; analysis of interactions at the crystallographically observed monomer interfaces identified interactions between chains A-B and C-D (Extended Data Fig. 1 b) possibly reflecting dimeric HSE in solution (Extended Data Fig.1 c, d). The structure reveals a stereotypical αββα MBL- fold with two central mixed β-sheets (I and II), comprised of 8 and 5 strands respectively, surrounded by helices (Fig. 1 a). The metal containing active site is adjacent to the dimer interface (Extended Data Fig 1 b) rationalizing reduced dimerization as manifested by metal ligand substitution (D120K) or metal removal (Extended Data Fig. 1 c, d, e). HSE has four of the five characteristic MBL metal binding motifs, His1 16, His1 18 Asp120 and His121 (motif II), His196 (motif III), Asp221 (motif IV) and His263 (motif V) (Fig. 1 b), (using BBL numbering) with two waters completing coordination. In the structure the active site of recombinant HSE produced in E. coli was assigned with two iron ions (Fig. 1 b), though HSE produced in HEK293 cells preferentially binds zinc ions (Extended Data Fig 1f).

Comparison of the HSE structure with those of other MBL-fold proteins reveals it is part of the glyoxalase II MBL structural subfamily (Fig. 1 c) (Extended Data Fig 2a, b). Although there is low sequence similarity (27 %), HSE is structurally similar to the human endoribonuclease β- lactamase-like-protein 2 (LACTB2), RMSD 2.23 A over 153 Ca atoms (Fig 1 c), an endoribonuclease responsible for mitochondrial imRNA maturation. Comparison of the HSE and LACTB2 active sites reveals near identical di-metal ion binding modes and immediate active site residues (Extended Data Fig. 2a). Two loops in close proximity to the HSE active site (β3-β4 and β14-α3 loops) are also present in LACTB2 (β1 -β2 and β1 1 -α3 loop) (Fig. 1 c), supporting a possibly similar substrate recognition/catalysis mechanism. Studies on the 'true' MBLs involved in beta-lactam antibiotic resistance, imply the two active site loops are likely involved in HSE substrate recognition. Two other regions in the HSE structure, (aa 51 -66 and the C-terminal region, aa 239-266) are disordered implying flexibility and possible involvement in substrate recognition. LACTB2 is reported to have high overall structural similarity with CPSF73. However, whilst comparison of the HSE with CPSF73 MBL domains reveals similar di-zinc ion binding modes, there are clear differences in the active site loops; only the β3-β4 HSE active site loop (β1 -β2 in LACTB2), is present in CPSF73 (β1 -β2 loop), where it is relatively shorter (Extended Data Fig. 2c).

Overall, the structural analyses suggested that HSE could be a ribonuclease, related to LACTB2, but with a different substrate type. We therefore purified recombinant wildtype HSE (wtHSE) and the D120K variant (D120K HSE) (which disrupts zinc binding motif 2 as reported for other MBL-fold proteins) from HEK293 cells and tested them as ribonucleases using a [a- 32 P]UTP labelled exogenous RNA substrate (Fig. 1 d). wtHSE displayed clear ribonucleolytic activity degrading most of the substrate under the standard assay conditions; D120K HSE manifested <50% of wtHSE activity. Since HSE catalysed RNA degradation led to the observation of some relatively large RNA fragments, like CPSF73, isolated HSE has substantial sequence-independent endoribonuclease activity. In agreement with this, no single nucleotide accumulated under any of the tested condition (data not shown). Overall, consistent with the structural similarity observed between the HSE and LACTB2, these results assign HSE as a ribonuclease with endonucleolytic activity.

We then investigated the subcellular localization of HSE. In HeLa cells, recombinant HSE showed near exclusive nuclear localization, especially to the nuclear envelope (NE) (Fig. 1 e, upper panels), supported by co-localization of HSE with lamin B1 (Fig. 1 e, lower panels). Although HSE showed a similar nuclear distribution pattern as the nucleoporins NUP153 and NUP214 (localized on the inner and outer NE membranes, respectively), no evident co- localization was observed, suggesting that HSE is a NE associated protein, but likely not part of the nuclear pore complex (Extended Data Fig. 3a). Transmission electron microscopy analyses in HeLa cells supported the localization of HSE to the NE in the vicinity of the inner nuclear membrane, though some HSE was also within the nucleus (Fig. 1f) (Extended Data Fig. 3b). The combined structural, localization, and catalytic observations defined HSE as a nuclear restricted MBL-fold endonuclease, potentially involved in RNA maturation.

Given the established role of CPSF73 as the nuclear ribonuclease responsible in pre-mRNA 3' end maturation, we investigated such a role for HSE. In support of this, co- immunoprecipitation experiments in HEK293 cells revealed an interaction between recombinant HSE and polyribonucleotide 5'-hydroxyl-kinase Clp1 (CLP1 ), which is a component of the pre-mRNA cleavage complex II (CF-II) involved in mRNA 3'end formation (Extended Data Fig. 3c), as identified by the Biological General Repository for Interaction Datasets (BioGrid). HSE was then depleted in HeLa cells using siRNA as, for comparison, was CPSF73 (Fig. 2a). To evaluate not only the polyadenylated mRNA population (polyA+), but also RD histone mRNA (SL) processing, cells were synchronized in early S-phase. Chromatin RNA-seq (ChrRNA-seq) analyses for model genes (polyA+ mRNA), e.g. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), displayed clear transcription termination defects downstream of the transcription end site (TES) in CPSF73, but not HSE, depleted cells, nor controls (Fig. 2b). Unexpectedly, RD histone genes (SL mRNA) showed substantial transcription termination defects, not only in CPSF73, but also in HSE, knockdowns (Fig. 2c). Importantly, meta-analyses on 46 not-overlapping RD histone gene manifested strong transcription termination defects only in the HSE depleted samples. Thus, read through (RT) transcripts corresponding to this defect were clearly detectable covering ~ 400 bases downstream of the TES (Fig. 2d).

Quantitative reverse transcription PCR (RT-qPCR) analyses validated the transcriptomics results. With normal protein-coding mRNA (polyA+), transcription termination was substantially affected by CPSF73, but not HSE, depletion (Fig. 2e). The abundance of termination defective (3' end extended) RD histone mRNA increased in both HSE and CPSF73 depleted cells compared to controls (Fig.2e), leading to the assignment of HSE as a RD histone selective pre-mRNA processing endonuclease.

We next used flow cytometry to investigate the role of HSE in cell cycle progression given the strong connection between histone biosynthesis and DNA replication (Fig. 3). Unsynchronized cells depleted for either HSE or CPSF73 manifested increased accumulation in G-i/early S- phase and decreased proportions of cells in G2 compared to controls. Thymidine treated cells harvested immediately after block release showed the expected G-i/early S phenotype. However, four hours post release, controls normally progressed through the cell cycle, while HSE and CPSF73 depleted cells manifested a strong delay in Gi/early S-phase. Notably CPSF73 depletion showed a small population of G2 cells 4h post release, not observed with HSE depletion (Fig. 3a). Western blots were then used to investigate the nature of the delay in cell cycle progression by monitoring levels of cell cycle protein markers 31 (Fig. 3b). Cyclin D1 (a Gi marker) was hardly detectable in controls, but upregulated on HSE depletion and to a lesser extent with CSPF73 depletion. These results are consistent with the observation of a relatively stronger Gi phenotype with HSE, compared to CPSF73, depletion. Both HSE and CPSF73 depletions showed higher cyclin E (a d/S boundary and S progression marker) expression level compared to controls following thymidine block, supporting a cell cycle progression delay. The relative cyclin D1/E levels reflect the flow cytometry analyses where both depletions cause delay in cell cycle progression (Fig.3a, b).

To investigate the role of HSE in S-phase, incorporation of 5-bromo-2'-deoxyuridine (BrdU) into newly synthesized DNA in synchronized cells was monitored by flow cytometry (Fig. 4a, upper panels) followed by propidium iodide (PI) staining (Fig. 4a, lower panels). The abundance of BrdU positive cells, corresponding to S-phase cells, was strongly reduced in HSE and CPSF73 knockdowns at early time points, (30 minutes, 1 hour) compared to controls (Fig. 4a, upper panels; Fig. 4c). Later time points displayed a reduced difference, though 6 hours post release a consistent pool of Gi cells was still apparent in HSE depleted cells compared to both control and CPSF73 depleted cells (Extended Data Fig. 4). These results reveal both HSE and CPSF73 depleted cells are strongly impaired in efficiently entering/progressing in S-phase. Although both depletions displayed a strong delay 30 minutes post release, the defect was more pronounced with HSE depletion over the full cell cycle, implying a more important cell cycle specific role for HSE (Extended Data Fig. 4). In control samples (siLuc) HSE levels were increased 6 hours post release (Fig. 4d) corresponding to mid S-phase (as shown by flow cytometry profiles, Figure 8) supporting a specific role for HSE in S-phase. By contrast, CPSF73 expression levels were constant (18 hours), consistent with its broad role in pre-mRNA processing (Fig. 4d). Although both HSE and CPSF73 depleted cells progress through the complete cell cycle, levels of cyclins D1 and E were strongly modified compared to controls as discussed above. Histone H3 levels were used to analyse RD histone protein abundance in HSE and CPSF73 depleted cells. H3 levels were strongly impaired in HSE depleted cells; by contrast CPSF73 knockdown showed an intermediate H3 level compared to controls. An even more severe phenotype was observed in a HSE stable knockdown generated in HeLa cells by clustered regularly-interspaced short palindromic repeats (CRISPR/Cas9) technology (Fig. 4b, c, e) (Extended Data Fig. 6) supporting a specific role for HSE in cell cycle regulation, in particular on entering/progressing through S-phase (Fig. 4b, c). Like the siRNA mediated knockdown, CRISPR/Cas9 depletion of HSE results in an altered production of cyclin E and reduced level of H3. By contrast cyclin D1 levels appear unchanged, possibly due to a mechanism of cellular adaptation under unfavourable conditions (Fig. 4e).

Depletion of either HSE or CPSF73 caused differential regulation of four genes (TGFB2, CCND1 , MY016, and TGFBR1 ) known to be directly involved in cell cycle arrest in d or in the Gi/S-phase transition (Fig. 4f). HSE depletion was also observed to cause differential regulation of two other genes (TP63 and PIM1 ) involved in cell cycle progression, which were not differentially regulated with CPSF73 depletion (Fig. 4f), supporting a cell cycle specific role of HSE (Extended Data Fig. 5a).

The combined biochemical, structural, genetic, and cellular studies define HSE as an endoribonuclease specific for 3 ' end processing of RD histone pre-mRNA during S-phase. The slow grow rate of cells lacking HSE likely reflect its important role in RD histone pre- mRNA processing which is required for a normal progression through the cell cycle. Although at least during S-phase, HSE has the major and specialized role in RD histone pre-mRNA processing, consistent with previous reports, we found that CPSF73 can also promote RD histone pre-mRNA processing. It is possible that CPSF73 is relatively more important in RD histone pre-mRNA processing outside of S-phase, as it does for polyadenylated pre-mRNA sequences, including for multiple histone variants. Future work can focus on defining the factors targeting HSE to the RD histone pre-mRNA3 ' cleavage site (Extended Data Fig. 5b). Inhibiting HSE may act to selectively slow S-phase progression in cancer cells and so could represent a new type of S-phase specific cancer target. True bacterial MBLs involved in antibacterial resistance have been shown to be amenable to small molecule targeting and though extensive work will be required, this is also likely to be the case for the human MBL- fold endonucleases. Our results suggest that targeting HSE may not be as toxic as CPSF73 which, owing to its pleiotropic role in polyadenylated pre-mRNA processing, is essential. Finally, it is notable that relatively non-selective zinc ion chelating compounds, including HDAC inhibitors such as SAHA (suberanilohydroxamic acid), are already used for cancer treatment via S-phase targeting. It is possible that these, at least in part, work by inhibiting endoribonucleases, such as HSE.

HSE shows endoribonucleolytic activity in vitro

Overall, the structural, localization and genetic analyses suggested that HSE could be a ribonuclease, specifically involved in histone pre-mRNA processing. We therefore purified recombinant wildtype HSE (wtHSE) and a H196A/ D221A/ H263A variant (MUT HSE) (which alters binding of both zinc ions in the active site) from E. coli and tested them as ribonucleases using a [a-32P] UTP labelled RNA substrate corresponding to a fragment of the HIST2H3C gene (Fig. 13a, b). wtHSE displayed clear ribonucleolytic activity degrading the substrate in a time dependent manner under the standard assay conditions; mutHSE manifested substantially no activity over time. Since HSE catalysed RNA degradation led to the observation of some more abundant RNA fragments, we investigated the potential cleavage specificity of HSE on histone substrates. A modified RNA substrate (MUT RNA) consisting in a single nucleotide substitution (A/G) at the preferential histone pre-mRNA processing site was used in comparison to the unmodified substrate (WT RNA) used in the previous experiments (Fig. 13b). Both, wt and mutRNA substrates were either [γ-32Ρ] ATP 5' or [a-32P] UTP internally labelled to evaluate fragments size and their 5' to 3' orientation. wtHSE displayed ribonucleolytic activity on the wtRNA substrate degrading it in a time dependent manner as previously observed while surprisingly no ribonucleolytic activity was observed when wtHSE was incubated in presence of the modified RNA substrate (Fig. 13c). Furthermore, early detectable degradation products corresponded to the two fragments generated by RNA cleavage in correspondence of the CA dinucleotide (82 and 86 nt, 5' a 3' fragments, respectively) specific for histone pre-mRNA processing. Overall, consistent with the structural similarity observed between the HSE and LACTB224, these results assign HSE as a ribonuclease with endonucleolytic activity. In agreement with this, no single nucleotide accumulated under any of the tested condition (data not shown). Unlike CPSF736, that exhibit substantial sequence-independent endoribonuclease activity, isolated HSE has clear sequence-specific endonucleolytic activity possibly identifying the CA cleavage site as the initial cleavage site followed by further fragments degradation.

Loss of HSE can lead to the production of polyadenylated histone mRNAs and to inefficient transcription termination

As expected, single gene analyses on chrRNA confirmed the increased abundance of termination defective (3' end extended) RD histone mRNA in HSE CRISPR/Cas9 mediated knockdown in comparison to wildtype HeLa cells after synchronization in early S-phase (Fig. 1 1 a, extended Fig. 7a), further supporting the observed cell cycle phenotype. To investigate the nature of the defective transcripts, total RNA extracted from early S-phase synchronized HeLa cells (wildtype and the HSE CRISPR/Cas9 mediated knockdown) was used to evaluate the accumulation of defective histone pre-mRNAs in either the polyadenylated (poly-A plus) or unpolyadenylated (poly-A minus) RNA cellular fractions (Fig.1 1 b, extended Fig. 7b). Quantitative reverse transcription PCR (RT-qPCR) analyses showed an increase of about 15 % of histone transcripts in the poly-A plus fraction in HSE depleted cells compared to wildtype cells; at the contrary, in both wildtype and HSE depleted cells the poly-A minus fraction showed a more similar abundance of histone transcripts (or a lower abundance in the HSE depleted cells in few analysed genes). The starting total RNA abundance of the analysed histone genes was also evaluated in both wildtype and HSE depleted cells to ensure the presence of a similar transcripts abundance before the poly-A plus/minus selection. Further GAPDH and the ribosomal 18S RNAs were used as controls to confirm the poly-A plus/minus selection efficiency. Our findings may suggest, as reported in previous studies, that histone mRNA may undergo through polyadenylation as normal protein-coding genes when improperly processed. ChIP experiments were then performed to further investigate the processing and consequent termination defect due to the loss of HSE in early S-phase synchronized HeLa cells. Interestingly, RT-qPCR analyses highlighted a significant increased abundance of RNA polymerase II (Pol II) on histone gene bodies (GB) (5' gene region) in HSE depleted cells compared to wildtype HeLa cells supporting the supposed transcription termination defect due to inefficient pre-mRNA cleavage. As expected the abundance of Pol II on the 3' end region (read though, RT) of the tested histone genes was reduced in both wildtype and HSE deplete cells compared to the abundance on the 5' gene region. However, HSE depleted cells showed an appreciable higher amount of Pol II on the 3' end region compared to wildtype cells supporting the observed transcripts extension likely due to inefficient pre-mRNA processing. RT-qPCR analyses also showed a moderate increase of CPSF73 on the 3" end region of some of the tested histone genes compared to the abundance in the 5' gene region as expected . Interestingly, a moderate increase of CPSF73 on the 3' end region of some of the tested histone genes was observed in HSE depleted cells compared to wildtype HeLa cells. This observation could be a preliminary indication of a possible functional redundancy between HSE and CPSF73, as well as consequence of a slower transcription rate or a direct consequence of the higher abundance of Pol II on histone genes due to the loss of HSE; further investigations will be required to confirm any of these hypotheses.

Small molecule HSE inhibitors reduce the level of histone H3 in vivo

The inventors have generated two small molecule inhibitors of HSE activity, designated 382 and 474, both of which inhibit HSE activity in cell-free conditions. The ability of these inhibitors to inhibit cellular HSE was investigated by treatment of HeLa cells.

HeLa cells were synchronized using the double thymidine block method. In brief, the cells were treated with 2 mM thymidine (final concentration) for 18 hours; the thymidine was then removed for 9 hours, and then was added again (2mM) for 15 hours. Cells were washed with PBS twice, and incubated in presence of the compounds (or only DMSO in control samples, CTR) for 24 hours. Cells were then harvested and cell lysates were used in western blot analysis to evaluate the protein level of histone H3, β-actin used as loading control.

The results are shown in Figure 12, where it can be seen that H3 levels are decreased (indicative of HSE inhibition) only in the samples treated with the compound 474. Without wishing to be bound by any hypothesis, the inventors believe that compound 382 is not able to enter the cell in a manner that allows it to inhibit cellular HSE activity.

Methods

Reagents and antibodies

Unless otherwise specified, reagents were from Sigma-Aldrich. Antibodies used were as follows: goat anti- HSE (sc-243427), HRP-conjugated donkey anti-goat (sc-2020), goat anti- Lamin B1 (sc-30264), mouse anti-Cyclin D (sc-20044), mouse anti-Cyclin E (sc-247) (all from Santa Cruz Biotechnology, Inc.); mouse anti-His tag (ab18184), rabbit anti-His tag (ab9108), rabbit anti-CLP1 (ab133669), rabbit anti-NUP214 (ab70497), mouse anti-NUP153 (ab24700), rabbit anti-Histone H3 (ab10799), HRP-conjugated mouse anti- -actin (ab49900) (all from Abeam); HRP-conjugated goat anti- rabbit (170-6515) (Biorad); rabbit anti-CPSF73 (A301 - 091 A) (Bethyl Laboratories, Inc,); HRP-conjugated goat anti- mouse (W402B) (Promega Corporation); FITC mouse anti-BrdU (364104) (BioLegend, Inc.). Alexa fluoi ® secondary antibodies used in immunofluorescence microscopy experiments were from Molecular Probes (Thermo Fisher Scientific). Recombinant HSE production in E. coli and purification

cDNA (codon optimized for expression in E. coli) encoding for HSE {GeneART ® , Thermo Fisher Scientific) was inserted into the pCOLD I vector (Addgene) to produce HSE with an N- terminal hexa-histidine tag (6xHis) with an A/-terminal 3C human rhinovirus (HRV3C) protease cleavage site. Recombinant HSE protein was produced in E. coli BL21 (DE3) cells grown in 2TY growth media with 50 pg/mL ampicillin at 37°C to mid-exponential phase (OD 600 = 0.6- 0.8). HSE production was induced by addition of isopropyl β-D-l -thiogalactopyranoside (IPTG) (0.5 mM) supplemented with 50 μΜ zinc sulfate while incubating at 15°C. Cells were harvested by centrifugation (6500 x g, 8 min) after 18h and frozen in liquid nitrogen. The cell pellet (-20 g) was added to 100 ml_ of lysis buffer, (20 mM 4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid (HEPES), pH 7.5, 500 mM sodium chloride, 5 mM imidazole), lysed by sonication followed by centrifugation (20,000 x g, 20 min). HSE was purified by loading the supernatant onto a 5 mL Ni-affinity column (GE Healthcare). The equilibration buffer was the same as the lysis buffer. The elution buffer additionally contained 500 mM imidazole and was used to form an imidazole gradient (from 5 to 500 mM) to elute the His- tagged protein. HSE was then loaded onto a S200 (300 mL) gel filtration column using 20 mM HEPES, pH 7.5, 500 mM sodium chloride as running buffer. Fractions containing protein were analyzed by SDS-PAGE. The 6x-His tag was cleaved by addition of HRV3C protease (overnight incubation at 4°C), then purified by Ni-affinity chromatography to remove the cleaved tag. The resultant purified HSE, (-27 kDa), was buffer exchanged into 25 mM HEPES, pH 7.5, 30 mM sodium chloride, then concentrated to 23 mg/mL using a Centricon concentrator (10k MW cutoff) (Merck) (at 3,000 x g) until the desired volume was achieved.

Crystallization and structure determination

Crystallization was performed using the sitting drop vapor diffusion method using Art Robbins 96 well - 3 subwell Intelliplates ® and a protein:well drop size of 200 nl_:100 nl_,100 nL:100nl_, 100 nl_:200 nl_ for each subwell condition. HSE crystallized over -24 hours using the following conditions: JCSG-p/us condition 94, 0.1 M Bis Tris pH 5.5, 0.2 M ammonium acetate, 25 % w/v PEG 3350 (protein to reservoir ratio 2:1 , 1 :1 , and 1 :2), (Molecular Dimensions). Crystallization conditions were optimized varying the ammonium acetate concentration (0.14 - 0.25 M) and PEG 3350 percentage (23-30 %). The resultant crystals, (-150 x 300 pm), obtained in 0.1 M Bis Tris pH 5.5, 0.18 M ammonium acetate, and 25 % w/v PEG 3350, were cryo-protected in well-solution diluted to 12.5% sucrose for 30 seconds, then harvested using nylon loops followed by cryo-cooling and storage under liquid nitrogen. Data were collected on a single crystal at 100 K at the Diamond Light Source Synchrotron (beamline I04, 1 .28268 A wavelength) to 1.8 A resolution. Data were autoprocessed at the beamline using XDS 34 and CCP4-SCALA 35 in XIA2 36 . The HSE structure was solved by molecular replacement (MR) using the PHASER subroutine within PHENIX 37 39 with an ensemble structure as a search model based on 1 1 crystal structures identified by the Phyre 2 modelling server 40 using the HSE protein sequence as search input. Refinement was carried out by iterative rounds of model building using Coot 41 and maximum likelihood restrained refinement using PHENIX 42 . Ramachandran statistics calculated 98.27 % most favored geometry, 1 .61 % additionally allowed and 0.12% outliers. Data collection, processing, and structure refinement statistics are given in Extended Data Table 1 .

Non-denaturing electrospray ionization mass spectrometry

HSE purified from E. coli was diluted to 15 μΜ in 15 mM ammonium acetate buffer (pH 7.5). The cone voltage for the acquisition of the spectra was 80 V. Electrospray ionization mass spectra of the apo-enzyme were acquired in the positive ionization mode after overnight metal removal treatment by addition of 20 mM ethylenediaminetetraacetic acid (EDTA) to the protein solution.

Inductively coupled plasma mass spectrometry (ICP-MS)

WT and D120K HSE were produced in E. coli or HEK293 cells were purified, 30 i of the resultant purified protein was then buffer exchanged into 20 mM HEPES pH 7.5, 50 mM NaCI buffer prepared using ultrapure water. Concentrations of Zn, Fe, Mn, Ni, and Co divalent ions were measured in triplicate. ICP-MS experiments and data analysis were carried out by the ICP-MS Trace Element Small Research Facility of the Earth Sciences Department, Oxford University.

Cell culture and transfection

HeLa {ATCC) and HEK293 (kindly provided by Prof. Peter McHugh) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and 2 mM L-glutamine, at 37°C in a humidified incubator (5% CO2). To generate the construct HSE 6xHis-pCDNA 3.1 , a sequence coding for HSE (GeneART ® , Thermo Fisher Scientific) was amplified by PCR to introduce a C-terminal 6xHis tag, then cloned into the pCDNA 3.1 vector (Invitrogen). Both HeLa and HEK293 cells were transiently transfected with the HSE 6xHis- pCDNA3.1 construct using the Fugene ® HD transfection reagent {Promega), following the manufacturer's instructions. HSE production was monitored by western blotting, 24 and 48 hours after transfection. To generate a stable cell line overexpressing HSE, HEK293 cells were transfected with the HSE 6xHis-pCDNA 3.1 construct using the Fugene ® HD transfection reagent {Promega), following the manufacturer's instructions. Stably transfected cells were selected by addition of geneticin (G418) (1 mg/ml) to the cell culture media; cell clones were generated by limiting dilution plating. Clones were analyzed by western blotting for their capacity to produce HSE.

HEK293 expressed HSE production and purification

About 70 x 10 6 of HEK293 cells overexpressing the HSE protein (clone 27, c27) were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Tris-HCI, pH8, 150 mM NaCI, 1 % Triton X-100, 0.1 % SDS, 0.5% sodium deoxycholate, 1x protease inhibitor cocktail). The lysates were then centrifuged (14,000 x g, 10 minutes, 4 °C). The supernatant containing total cellular proteins was diluted 1 :1 (v/v) in phosphate buffer saline solution (PBS), with 10 mM imidazole and about 100 μΙ_ of Ni-NTA resin (Biorad) and incubated for 2 hours at 4°C with stirring. The resultant resin was washed with PBS containing 10 mM imidazole (5 times), followed by 3 more washes with PBS containing 50 mM imidazole. HSE was eluted with the same buffer containing 400 mM imidazole. The solution was concentrated to about 100 μΙ_ using a Centricon concentrator (10k MW cutoff) (Merck) and exchanged into 25 mM HEPES pH 7.5, 50 mM NaCI using 0.5ml_ Micro Biospin™ 6 desalting columns (Biorad). The solution was concentrated to about 0.5 mg/mL and purity determined by SDS PAGE.

Single point mutagenesis

Site-directed residue substitutions of HSE were performed using Pfu Turbo DNA polymerase (Agilent Technologies) according to the manufacturer's protocol. Primers used to generate the mutants were designed following the manufacturer instructions and are listed in Extended Data Table 2. The pCOLD I and pCDNA 3.1 vectors (used to overexpress HSE in E. coli and HEK293 cells, respectively) were employed as template DNA. Plasmid DNA with the correct mutation were used to transform E. coli BL21 (DE3) cells or to stably transfect HEK293 cells as described above. HSE mutants expressed in both E. coli and HEK293 cells were produced and purified similarly to the wild type enzyme as previously described.

In vitro RNA degradation assays

A 300 nucleotide DNA sequence coding for a portion of the yeast phosphoglycerate kinase 1 (PGK1 ) gene with a 5' T7 RNA polymerase promoter sequence was used as a template to generate an internally [ 32 P] labelled RNA substrate by in vitro transcription (MEGAscript ® T7 Kit, Thermo Fisher Scientific). The reaction was carried out following the manufacturer's instructions by adding an excess of [a- 32 P] UTP to the reaction mixture. In vitro transcription products were fractionated on a 6% denaturing polyacrylamide/urea gel. The successful generation of the radiolabeled RNA substrate was monitored by autoradiography. The radiolabeled RNA was then gel purified and resuspended in 50 μΙ_ ddH20. Cleavage assays were carried out in a reaction mixture (10 μΙ) containing 1 μΙ_ of labelled RNA substrates, 300 ng of recombinant HSE produced in HEK293 cells and 1x reaction buffer (20 mM HEPES KOH pH 7.5, 50 mM KCI, 10 mM MgCI 2 , 0.5 mM DTT, 0.05 % Triton X-100, 5 % glycerol) at 37°C. The reaction was stopped at the appropriate time point by heating the samples (5 minutes, 70 °C), in the presence of formamide loading dye. RNA degradation products were then fractionated on 6% denaturing polyacrylamide/urea gel and the data were analyzed by Phospho-lmager (Fujifilm FLA 5000 imager). Densitometric evaluation of the intact RNA substrate was carried out using AIDA image analyzer software (Elysia-raytest GmbH).

Alternative in vitro RNA degradation assay method:

A 168 nucleotide DNA sequence coding for a portion of the human histone 2H3C (HIST2H3C) gene with a 5' T7 RNA polymerase promoter sequence was used as a template to generate a 5' or internally [ 32 P] labelled RNA substrate by in vitro transcription (MEGAscript ® T7 Kit, Thermo Fisher Scientific). For the internally labelled substrate the reaction was carried out following the manufacturer's instructions by adding an excess of [a- 32 P] UTP to the reaction mixture. In vitro transcription products were fractionated on 8 % denaturing polyacrylamide/urea gel. The successful generation of the radiolabeled RNA substrate was monitored by autoradiography. The radiolabeled RNA was then gel purified and resuspended in 80 μΙ_ ddhbO. Alternatively, the 5' [y- 32 P] ATP labelled substrate was obtained as follows: the same RNA template coding for a portion of the human HIST2H3C gene was produced by in vitro transcription. The obtained RNA was first digested with TURBO DNAse followed by alkaline phosphatase (Thermo Fisher Scientific) treatment. The obtained RNA was phenol- chloroform extracted and ethanol precipitated. Totally 10 pg of RNA were treated with T4 polynucleotide kinase (NEB) in presence of an excess of [γ- 32 Ρ] ATP. The obtained 5' [y- 32 P] ATP labelled substrate was then gel purified as above and resuspended in 50 μΙ_ ddh^O. The same portion of the human HIST2H3C gene carrying a single point mutation (A/G) at the cleavage site required for histone RNA maturation was produced and labelled as above. Cleavage assays were carried out in a reaction mixture (10 μΙ) containing 1 μΙ_ of labelled RNA substrates ( ~ 600 ng of [a- 32 P] UTP substrate, ~ 120 ng [y- 32 P] ATP substrate), 500 ng of recombinant wild-type or the active site variant HSE produced in E. coli and 1x reaction buffer (20 mM HEPES KOH pH 7.5, 50 mM KCI, 10 mM MgCI 2 , 0.5 mM DTT, 0.05 % Triton X-100, 5 % glycerol) at 37°C. The reaction was stopped at the appropriate time point by phenol- chloroform extraction and ethanol precipitation. RNA degradation products were then fractionated on 8 % denaturing polyacrylamide/urea gel and the data were analyzed by Phospho-lmager (Fujifilm FLA 5000 imager).

Immunofluorescence microscopy HeLa cells transiently transfected with the HSE 6xHis-pCDNA 3.1 expressing construct were grown on glass coverslips for 24 hours after transfection, then briefly washed with PBS, fixed in PBS containing 3 % paraformaldehyde for 20 minutes at room temperature, then permeabilized in PBS containing 0.1 % Triton X-100 for 4 minutes at room temperature. Fixed cells were blocked in PBS containing 0.2 % fish skin gelatin (FSG) for 30 minutes at room temperature. Immunofluorescence staining was performed by incubating coverslips with the appropriate primary antibody diluted in PBS containing 0.2 % FSG, followed by the specific Alexa fluor ® 488 or 635 conjugated (Thermo Fisher Scientific) secondary antibody 43 . All primary antibodies incubations were carried out overnight at 4 °C. Secondary antibodies diluted in PBS containing 0.2 % FSG were incubated for 30 minutes in the dark. For 4", 6- diamidino-2-phenylindole, dilactate (DAPI) staining, coverslips were incubated for 2 minutes in PBS containing 0.2 % FSG using a 1 :500000 dilution from DAPI stock. Images were obtained at the Micron Advanced Bioimaging Unit (University of Oxford, UK) using an invert DVcore [Olympus) fluorescence microscope using a 100X/1 .35 objective. Image acquisition and deconvolution were performed using Resolve3D SoftWoRx-Acquire (Version 5.5.1 , Relase 3, Applied Precision Inc, Issaquah, Washington). Images were then analysed using the Fiji-Image J software .

Transmission electron microscopy

HeLa and HEK293 cells were transiently transfected with the HSE 6xHis-pCDNA 3.1 construct; cells were cryo-fixed 24 h post transfection and immunolabelled using a mouse anti- His tag antibody and a goat anti-mouse conjugated to 10 nm colloidal gold. Sample preparation and immunogold cryo-electron microscopy experiments were carried out at the Dunn School Electron Microscopy Facility, Oxford University. siRNA transfection and cell synchronization

About 50 % confluent HeLa cells were transfected with a SMARTpool of siRNA for HSE or CPSF73 (GE Dharmacon), or with a negative control siRNA (Luciferase), using the Lipofectamine™ RNAiMAX transfection reagent (Invitrogen) following the manufacturer's instructions. Cells were transfected with a final concentration of 10 nM siRNA and knockdown efficiency was assessed 48 hours post transfection by western blot analysis. Transfected HeLa cells were synchronized using the double thymidine block method 45 . In brief, 6 hours post siRNA transfection, the cells were treated with 2 mM thymidine (final concentration) for 18 hours; the thymidine was then removed for 9 hours, and then was added again (2mM) for 15 hours. Cells were then washed with PBS twice, and harvested at different time points after release from the block; alternatively, cells were washed with PBS twice and treated with 10 μΜ of 5-bromo-2'-deoxyuridine (BrdU), for 30 minutes or 1 hour. BrdU treated cells were washed with PBS to remove the unincorporated BrdU and then harvested at different time points for cell cycle analysis. Similarly, wt and CRISPR/cas9 mediated HSE kd HeLa cells were synchronized and BrdU treated as described above.

Cell cycle analysis

Synchronized HeLa cells were washed with PBS buffer and fixed in ice-cold ethanol overnight. DNA was stained with a propidium iodide (PI) solution containing 0.1 % Triton X-100 in PBS, 0.2 mg/ml RNase A, 0.02 mg/ml PI for 30 minutes at room temperature. Alternatively, BrdU treated cells were fixed by adding ice-cold 70% ethanol overnight. Cells were then incubated with 2 N HCI, 0.5% Triton X-100 for 30 min at room temperature and washed with 0.1 M sodium tetraborate buffer, pH 8 for 2 minutes. Cells were washed and incubated with a FITC conjugated anti-BrdU antibody (BioLegend) for 1 hour at room temperature. PI staining followed as described above. Cell cycle profiles were analyzed by flow cytometry with FACSCalibur (BD Biosciences) and processed using FlowJo software {Tree Star).

Chromatin RNA extraction and libraries preparation

Chromatin RNA (ChrRNA) was extracted from HeLa cells 48 hours after siRNA transfection to coincide with the end of the second thymidine block time-point. Approximately 3x10 6 cells were resuspended in 500 pL of ice-cold RLB buffer, (10 mM Tris-HCI, pH 7.5, 140 mM NaCI, 0.5% nonidet-P40 (NP-40), 1.5 mM MgCI 2 ), and lysed by adding an equal volume of RLB buffer with 24% (m/v) sucrose. Nuclei were collected by centrifugation (14,000 x g) at 4°C for 10 min. Isolated nuclei were resuspended in 120 μΙ of NUN1 buffer, (20 mM Tris-HCI, pH 7.9, 75mM NaCI, 0.5 mM EDTA, 50% Glycerol, 0.125 mM PMSF, 1 mM DTT), followed by addition of 1 .2 ml NUN2 buffer, (20 mM HEPES KOH pH 7.6, 7.5mM MgCI2, 0.2 mM EDTA, 300 mM NaCI, 1 M urea, 1 % NP-40, 1 mM DTT). Nuclei were incubated for 15 minute on ice with mixing by vortexing for 5 seconds every 5 min. Chromatin pellets were precipitated by centrifugation (14,000 x g) at 4°C for 10 min and then resuspended in 200 ml HSB buffer, (10 mM Tris-HCI, pH 7.5, 500 mM NaCI, 10 mM MgCI 2 ), in presence of 4 U TURBO DNase {Ambion) and incubated at 37°C for 20 minutes. Followed a 20 minutes proteinase K treatment (Roche) at 37°C, chromatin RNA (chrRNA) was phenol-chloroform extracted and ethanol precipitated. RNA was resuspended in 200 pL of 1x TURBO DNAse buffer in presence of 4 U TURBO DNase and incubated at 37°C for 30 minutes. The entire procedure of extraction, precipitation and DNase treatment was repeated twice. RNA was then extracted and precipitated again, prior to solubilisation in 75 pL of RNase free water.

Prior to RNA libraries preparation, rRNA was depleted using the Ribo-Zero Magnetic kit (lllumina) from 2.5 pg of ChrRNA. Libraries were prepared starting from 100 ng chrRNA using the NEBNext Ultra Directional RNA Library Prep kit for lllumina (NEB) following the manufacturer's instructions. Deep sequencing using Hiseq4000 with paired-end (75 bp) runs was performed by the Wellcome Trust Centre for Human Genetics (WTCHG), Oxford, UK.

Real time PCR analysis

ChrRNA was extracted from synchronized HeLa cells 48 hours after siRNA transfection as described above. For reverse transcription, 500 to 900 ng of ChrRNA from each sample was used to generate single-strand cDNA by incubation with random hexamers {Qiagen) and Superscript III reverse transcriptase (Life Technologies). Quantitative RT-PCR was performed using the SYBR Green Master Mix (Qiagen). Relative RNA levels were calculated using the ACt method. Data were acquired and analysed using Rotor-GeneQ (Qiagen). Defects in 3' end transcription termination were evaluated using amplicons located downstream of the 3' untranslated region (3' UTR) of RD histone genes. The relative abundance of unprocessed sequences was normalised to the relative abundance of an amplicon covering a portion of the GAPDH gene body (GB) or to the corresponding histone GB abundance. Primers used are listed in Extended Data Table 2.

Chromatin RNA-Seq data processing

GRCh38 was used as a reference genome. RNA-sequencing reads were trimmed using Cutadapt 1.8.3 46 . An in-house Perl scripts Perl script was used to remove the reads left unpaired. The remaining reads were then aligned to the human reference genome with Tophat 2.0.13 47 using parameters, tophat -g 1 -r 3000 -no-coverage-search. Aligned reads were processed to only include properly paired, properly mapped reads with no more than 2 mismatches using SAMtools 1.2 48 . Data were scaled to library size (genomeCoverageBed) using Bedtools 9 .For data vizualization, trackhubs for the UCSC browser were created by employing the UCSC bedGraphToBigWig tool 50 . Differentially expressed genes (siLUC vs. si HSE) and (siLUC vs. siCPSF73) were identified with Cuffdiff using default parameters.

Metagene profiles

Gene boundaries for histone genes and other protein coding genes were extracted from GRCh38, ENSEMBL gene annotation. Non-overlapping histone genes (TSS -200 bp, TES +500 bp) with an FPKM >1 were used to compute the metagene profiles. TSS and TES metagene profiles were obtained by plotting normalized read counts around annotated 3'end (TES plots) and 5' end (TSS plots) for sense strand relative to the direction of gene transcription. Graphs were plotted using Matplotlib 51 in Python 52 .

Co-immunoprecipitation analysis Nuclear lysates used in co-immunoprecipitation experiments were prepared as follows: HEK293 wild type and HEK293 stably producing the C-terminal his-tagged HSE (c27) cells were washed twice with ice-cold PBS, then suspended in harvest buffer (10 mM HEPES KOH pH 7.9, 50 mM NaCI, 0.5 M sucrose, 0.5 % Triton X-100, 0.2 mM DTT and protease inhibitor cocktail). After incubation for 5 minutes on ice, cells were centrifuged (100 x g 10 min at 4°C) to pellet nuclei. The supernatant containing cytosolic and membrane proteins was discarded and the nuclear pellet was resuspended and washed by centrifugation in buffer A (10 mM HEPES KOH pH 7.9, 10 mM KCI, 0.2 mM DTT and protease inhibitor cocktail) for 10 minutes at 100 x g at 4°C. The obtained nuclear pellet was resuspended in 4 volumes of buffer C (10 mM HEPES KOH pH 7.9, 200 mM NaCI, 0.1 % NP-40, 0.2 mM DTT and protease inhibitor cocktail) and vortexed at 4 °C for 30 minutes. The nuclear lysate was then centrifuged (15 minutes at 14,000 x g at 4 °C). The concentration of the obtained supernatant containing nuclear proteins was measured using the Bradford reagent. Co-immunoprecipitation experiments were carried out using the Pierce Co-lmmunoprecipitation (Co-IP) Kit (26149) (Thermo Scientific) following the manufacturer's instructions. About 250 μg of nuclear lysate from both HEK293 wild type and HEK293 c27 was diluted to 500 μΙ_ using the manufacturer's provided lysis/wash buffer and incubated with 50 μΙ_ of AminoLink Plus Coupling Resin pre- coupled with 10 μg of rabbit primary anti-his tag antibody {Abeam) for 2 hours at 4 °C with stirring. After centrifugation (1 ,000 x g , 1 minute) to remove unbound proteins, the resin was washed with the same lysis/wash buffer four times by centrifugation. Immunoprecipitated proteins were eluted using the manufacturer's acid elution buffer; co-immmunoprecipitates were resolved by 12% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare Life Science) for western blot analysis.

CRISPR/Cas9-mediated HSE deletion

Single guide RNA (sgRNA) constructs aimed to fully delete the HSE gene in HeLa cells were designed and produced by the Genome Engineering Oxford Facility, Oxford University, UK (Extended Data Table 2). HeLa cells were transiently co-transfected with two sgRNA constructs (vector epX459(1.1 )), both carrying the engineered hSpCas9(1 .1 ) gene 53 and a sgRNA cassette targeting the 5' or 3' region flanking the HSE gene using Fugene ® HD transfection reagent (Promega), following the manufacturer's instructions. 24 hours after transfection, cells were exposed to puromycin (2 μg/ml) for 24 hours to positively select transfected cells. After recovery, cells were harvested and used to select HSE depleted cell clones by limiting dilution plating and to isolate genomic DNA to evaluate the gene deletion efficiency by PCR (Extended Data Table 2). Genomic DNA was then extracted from cell clones and PCR reactions amplifying the DNA region flanking the sgRNA targeting sites were performed to confirm HSE gene deletion. PCR products corresponding to the wt gene or to the gene deletion were gel purified and their sequence was confirmed by Sanger sequencing. Total RNA was then extracted from cell clones carrying the HSE deletion using TRI reagent {Ambion) following the manufacturer's instructions and RT-qPCR analyses were performed to evaluate HSE mRNA level after CRISPR/Cas9-mediated depletion as above. RT-qPCR products were then analysed by Sanger sequencing.

Data analysis and statistics

The number of biological repeats and statistical tests (conducted in Microsoft Excel or GraphPad) are indicated in the corresponding figure legends. Error bars representing s.e.m. are shown where required.

Figures

Figure 1. HSE is a metallo β-lactamase fold endonuclease. (a) View from an HSE crystal structure (PDB ID: 4V0H) showing secondary structure elements and di-metal containing active site. Helices are in cyan, β-strands in yellow, and metal ions are orange spheres. Dashed line (in gray) indicates missing residues (aa 51 -66). (b) View of the metal binding and active site residues with representative electron density (3.0□ mFo-DFc OMIT; cyan mesh) for the side chains of His1 16 (Νε2 to Μ Ί : 2.33 A), His1 18 (Νε1 to Mi: 2.35 A), Asp120 (052 to M 2 : 2.9 A), His121 (Νε2 to M 2 : 2.37 A), His196 (Νε2 to Mi: 2.27 A) , Asp221 (052 to Mi: 2.14 A; 052 to M 2 : 2.12 A), His263 (Νε2 to M 2 : 2.06 A) and the two water molecules (red spheres) which coordinate (black dashed lines) to the metals (orange spheres). The BBL MBL numbering system is used 23 (active site motif number in parentheses 11 ). Numbering as observed in the structure are also shown in bold (c) Superimposition of HSE (cyan; metals in orange) and LACTB2 folds (PDB ID: 4AD9) 24 (pink; metals in grey), (d) wtHSE in vitro cleavage assay using a [ 32 P]RNA fragment (lanes 2 to 4) and D120K HSE variant (lanes 5 to 7). Densitometry of the intact RNA substrate is shown below; values are 10 4 unit scale, (e) HSE nuclear envelope localization. Upper panels, staining with mouse anti-His tag antibody (green) in combination with DAPI (blue). Lower panels, double staining with mouse anti-His tag (red) and goat anti-Lamin B1 (green) antibodies. Scale bar 10 μιη. (f) TEM analyses of HSE localization. Gold nanoparticles are arrowed. Abbreviations: C, cytoplasm, N, nucleus.

Figure 2. Loss of HSE leads to 3' end processing defects in RD histone pre-mRNA. (a)

Western blot showing the knockdown efficiency of siRNA treatments for HSE and CPSF73 in synchronized HeLa cells. β-Actin used as control, (b, c) ChrRNA-seq analyses of two biological replicates, (Set1 , Set2), on GAPDH or selected RD histone (HIST1 H2BC, HIST4H4) genes (UCSC Genome Browser 33 ) following HSE or CPSF73 depletion. Arrows indicate gene bodies and transcription termination defect orientation, (d) ChrRNA-Seq meta-profiles on RD histone genes following HSE or CPSF73 depletion. 46 non-overlapping RD histone genes were selected with two biological replicates, (e) Real Time-qPCR quantification of 3' end transcription termination defect following HSE and CPSF73 depletion on genes as in (b, c). Relative abundance (RA) of unprocessed RNA was normalized to GAPDH mRNA (black) or to the corresponding histone gene body abundance (white) (Extended Data Table 2). Error bars represent SEM from four biological replicates. Asterisks indicate the statistical significance observed using the two-tailed i-test (** P < 0.01 ). Unlabeled variations were considered not statistically relevant. Abbreviations: GB, gene body; RT, read through; TES, transcription end site.

Figure 3. Loss of HSE impairs cell cycle progression, (a) Cell cycle analyses of HSE and CPSF73 depleted cells. Analyses were on unsynchronized cells or after synchronization (using a double thymidine block) as indicated. Flow cytometry profiles were obtained by PI staining. Control siRNA (siLUC) transfected cells were used for reference, (b) Western blotting evaluating knockdown efficiency of HSE and CPSF73 after siRNA treatment, in unsynchronized and synchronized cells. Cyclin D1 (Gi marker) and E (Gi / S transition and S- phase progression marker) levels were analyzed. β-Actin was used as a control. Control siRNA (siLUC) transfected cells were used for reference.

Figure 4. Loss of HSE impairs normal entering to S-phase and its progression, (a) Cell cycle analyses of HSE and CPSF73 depleted cells after synchronization in early S-phase. (b) Cell cycle analysis of HSE CRISPR/Cas9 mediated stable knockdown (KD) cells after synchronization in early S-phase. Flow cytometry profiles obtained by a double-staining with an anti-BrdU antibody (upper panels) and PI (lower panels). Control siRNA (siLUC) transfected or wt HeLa cells were used for reference. Abbreviations: ES, early S-phase; i_S, late S-phase. (c) Comparative quantification of BrdU positive cells corresponding to ES cells in HSE and CPSF73 depleted cells and HSE KD cells after synchronization. Control siRNA (siLUC) transfected cells and wt HeLa cells were used for reference. Flow cytometry profiles were obtained by a double staining as before and cell populations were gated as in a, b (upper panels), based on the cell cycle stage. Error bars represent SEM from three independent experiments (biological replicates). Asterisks indicate the statistical significance observed using the two way ANOVA tool followed by the Bonferroni multiple comparison tool from Graphpad Prism 5 software: *, P < 0.05; **, P < 0.001 ; ***, P < 0.0001 . Unlabeled variations were considered not statistically relevant, (d, e) Western blot analyses (as in Fig. 3b) evaluating HSE CRISPR/Cas9 mediated stable knockdown efficiency and effects on cyclin D1 , E and histone H3 levels. Samples in e correspond to synchronized cells treated with BrdU (30 minutes incubation), (f) Differential expression analyses of Gi and Gi / S phase transition genes observed by ChrRNASeq. Asterisks indicate statistical significance: **, P < 0.001 ; ***, P < 0.0001. Calculated q-values for all analyzed genes were < 0.05.

Figure 5. Extended Data Figure 1. HSE is a functional dimer binding zinc ions belonging to the glyoxalase II MBL subfamily, (a) Clustal Omega 19 alignment of sequences for the MBL domains of HSE (SEQ ID NO:30), LACTB2 (SEQ ID NO:27), CPSF73 (SEQ ID NO:29), and glyoxalase II (SEQ ID NO:28). Secondary structure elements are derived from the HSE structure (PDB ID 4V0H). β-Sheets are shown as yellow arrows, a-helices as blue cylinders. Residues are colored based on conservation: dark blue represents the highest conservation grade, blue the second highest, light blue the third highest, and no color the least conserved. The conserved metal binding residues are indicated by a red asterisk (*) with the corresponding MBL motif is in parentheses, (b) HSE dimer model calculated by PISA 21 . Surface representation is shown for protomer A/C. The single amino acid variant D120K is shown (sticks), (c) Multi-angle laser light scattering (MALS) analyses of E. coli produced wt and D120K HSE. Peak A likely represents a trimer (-80000 Da); peak B (-54000 Da) a dimer; and peak C (-27000 Da) a monomer, wt HSE is predominantly dimeric, the D120K variant is a -1 :1 mixture of dimeric and monomeric states. This observation correlates with reduced activity of D120K HSE (main text Fig. 1 d), the proposal that the dimer is catalytically active, and the observation that the active site is close to the dimer interface. MALS experiments were carried out at the Biophysical Services of the Biochemistry Dept., Oxford University, (d) Non- denaturing PAGE analyses indicates wt HSE produced in either E. coli or HEK293 cells is predominantly dimeric in solution; a higher oligomeric state (trimer or tetramer) is also observed. HSE produced in either, E. coli or HEK293 cells shows the same oligomerization behavior, (e) Non-denaturing electrospray ionization mass spectrometry deconvoluted spectra of recombinant HSE produced in E. coli indicates that HSE is dimeric, binding two metal ions. Peak A (54880 Da) represents the dimer with 2 divalent transition metal ions (zinc or iron) bound to each monomer (+224 Da); peaks B and C (27440 and 54880 Da, respectively) correspond to monomer and dimer without bound metal, following metal removal using EDTA. (f) Inductively coupled plasma mass spectrometry (ICP-MS) experiments reveal that in human cells HSE binds zinc. Interestingly, the D120K variant was observed to lose -70% of the metal content of the wildtype, consistent with the partial loss of dimeric assembly for D120K HSE. Overall, these observations support the direct involvement of the active site metal ions in generating the catalytically active dimeric form.

Figure 6. Extended Data Figure 2. Structural comparison of the HSE with ribonucleases from the MBL superfamily and with glyoxalase II. (a) Superimposed active site residues from HSE (cyan) and LACTB2 (pink). Apparently identical residues in the proximity of the active site are shown. Zinc and iron ions are gray and orange spheres, respectively. The BBL numbering system for MBLs is used 23 .(b) Superposition of HSE (cyan), with glyoxalase II (PDB ID: 1 QH5) (brown) structures. Loops of interest are indicated. Detailed comparison, in particular of the loops surrounding the active site, supports the biochemical evidence that HSE is not a glyoxalase II; only one of these loops, (β10-α3 loop / glyoxalase I I; β14-α3 loop / HSE), is in glyoxalase II where it is in a shorter form. Zinc and iron ions are gray and orange spheres, respectively, (c) Superimposition of HSE (cyan) with CPSF73 (PDB ID: 2I7T) (gray). Note that only the MBL domain is used in the superimposition. Zinc and iron ions are gray and orange spheres, respectively.

Figure 7. Extended Data Figure 3. HSE localizes to the nuclear envelope and interacts with CLP1. (a) Immunofluorescence analyses of HSE subcellular localization visualized 24 h after transient transfection with the 6xHis-pCDNA 3.1 construct in HeLa cells. Upper panels, HeLa cells double-stained with rabbit anti-His tag (green) and mouse anti-NUP 153 (red) antibodies. Lower panels, HeLa cells double-stained with mouse anti-His tag (red) and rabbit anti-NUP 214 (green) antibodies. The merged images reveal similar subcellular distribution. Scale bar 10 μητι. (b) Transmission electron microscopic localization analyses of HSE in HEK293 cells; Gold nanoparticles (arrows) specifically locate HSE to the nuclear envelope, possibly the inner membrane, but more gold is also present within the nucleus compared to the prevalent nuclear inner membrane localization observed in HeLa cells. Abbreviations: C, cytoplasm, N, nucleus, (c) Nuclear lysates used in co-immunoprecipitation experiments were prepared from wt HEK293 (CTR) and HEK293 cells stably producing C-terminal 6xHis-tagged HSE (C27). Both lysates were incubated with 50 μί of AminoLink Plus Coupling Resin [Thermo Scientific) pre-coupled with 10 μg of rabbit primary anti-his tag antibody [Abeam). The efficiency of HSE immunoprecipitation was analysed by western blot using an anti-HSE antibody [Santa Cruz Biotechnology, Inc.). The interaction with CLP1 was analysed by western blotting using an anti-CLP1 antibody [Abeam). 0.1 % input (0,25 μg) and unbound proteins after precipitation (output) were used as controls.

Figure 8. Extended Data Figure 4. Loss of HSE impairs normal entering and S-phase progression. Cell cycle analyses of HSE and CPSF73 depleted cells after synchronization in early S-phase through a complete cell cycle (18 hours). Flow cytometry profiles obtained by a double-staining with anti-BrdU antibody (upper panels) and PI (lower panels). Control siRNA (siLUC) transfected cells were used for reference. Abbreviations: ES, early S-phase; i_S, late S-phase. Red arrows indicate Gi / early S cells 6h post release from the double thymidine block. Figure 9. Extended Data Figure 5. HSE is RD histone pre -mRNA processing endoribonuclease specifically involved in cell cycle regulation (a) ChrRNA-seq examples from two biological replicates (Set1 , Set2), showing upregulation of a cell cycle specific gene (CCND1 ) following depletion of HSE and CPSF73 proteins, as observed using the UCSC Genome Browser 33 . A positive control siRNA (siLuc) knockdown is shown. Arrows indicate gene bodies and transcription termination defect orientation. Note that a 3' end transcription termination defect occurs only in the CPSF73 depleted samples, (b) Schematic comparison of the previously reported complex targeting CPSF73 on RD histone pre-mRNA and a possible proposed complex for HSE.

Figure 10. Extended Data Figure 6. HSE CRISPR/Cas9 stable knockdown validation, (a)

PCR analyses (genomic DNA) evaluating the HSE gene deletion efficiency using the CRISPR/Cas9 system after clonal selection in HeLa cells. Primers used to validate the gene deletion are in Extended Data Table 2. Both full length gene (FL) and the deletion (DEL) PCR products were sequenced to confirm the PCR results, (b) RT-qPCR quantification (total RNA) of the relative abundance (RA) of mRNA encoding for HSE in WT and HSE CRISPR/Cas9 mediate KD HeLa cells confirms a substantial reduction of HSE mRNA level in the CRISPR/Cas9 mediate KD consistent with the observed protein level reduction (Fig. 4e). HSE mRNA level was normalized to GAPDH mRNA. RT-qPCR primers used to validate the HSE stable KD are in Extended Data Table 2. RT-qPCR products were then analysed by Sanger sequencing confirming HSE mRNA production in the CRISPR/Cas9 mediate KD. (c) PCR analyses (genomic DNA) confirming the incomplete deletion of the gene encoding for WT HSE in the CRISPR/Cas9 mediated KD as observed at mRNA (b) and protein level (Fig. 4e). Primers amplifying a portion of the HSE gene body and of its 3' flanking region were used to avoid PCR preferential amplification of the gene deletion present in the KD and are in Extended Data Table 2 listed as HSE_set1_F and CRISPR/Cas9_R. PCR products were sequenced to confirm the PCR results.

Figure 11. Extended Data Figure 7. The 3' end processing defect in RD histone pre-mRNA due to the HSE depletion increases the polyadenylated fraction of histones mRNA and leads to a transcription termination defect in HeLa cells, (a) Real Time-qPCR quantification of 3' end transcription termination defect in the HSE CRISPR/Cas9 mediated stable knockdown (KD) compared to wild-type (WT) HeLa cells. The relative abundance (RA) of unprocessed RNA was normalized to GAPDH mRNA (black) or to the corresponding histone gene body abundance (white) (Extended Data Table 2). Error bars represent SEM from four biological replicates. Asterisks indicate the statistical significance observed using the two-tailed t-test (*,P < 0.05, **, P < 0.01 , ***, P < 0.001 ). Unlabelled variations were considered not statistically relevant, (b) Real Time-qPCR quantification evaluating the enrichment of polyadenylated RD histone RNA transcripts in the HSE CRISPR/Cas9 mediated stable knockdown (KD) compared to wild-type (WT) cells. GAPDH and 18S rRNA were used as positive and negative controls, respectively. For each primer set, the amplicon abundance was calculated using the standard curve method and the relative percentage of transcripts in both, poly-A plus or minus fractions was expressed as ratios over the abundance in the total RNA used as starting material for the selection. The starting abundance of histone transcripts was also quantified in total RNA by the standard curve method to ensure similar histones RNA amount before the selection. Error bars represent SEM from two biological replicates Abbreviations: P, poly-A plus; M, poly-A minus, (c, d) ChIP analyses using antibodies to total RNA polymerase II (Pol II) and CPSF73 comparing wild-type (WT) and CRISPR/Cas9 mediated stable HSE knockdown (KD) HeLa cells after early S-phase synchronization. Columns represent average relative ChIP signal normalized to the maximal value observed for the antibody to RNA polymerase II. Error bars represent SEM from three biological replicates.

Figure 12. HeLa cells were synchronized using the double thymidine block method. In brief, the cells were treated with 2 mM thymidine (final concentration) for 18 hours; the thymidine was then removed for 9 hours, and then was added again (2mM) for 15 hours. Cells were washed with PBS twice, and incubated in presence of the compounds (or only DMSO in control samples, CTR) for 24 hours. Cells were then harvested and cell lysates were used in western blot analysis to evaluate the protein level of histone H3 (as used in the paper), β-actin used as loading control.

Figure 13. HSE has sequence specific endoribonucleolytic activity on RD histone pre-mRNA in vitro, (a) wtHSE time dependent in vitro cleavage assay using an internally labeled [32P] histone pre-mRNA fragment and an active site substituted HSE variant, (b) Schematic view of the histone 2H3C pre-mRNA fragment used in the cleavage assays showing the preferential cleavage site occurring after a CA dinucleotide located five nucleotides downstream of the stem loop. The single nucleotide substitution (A/G) generated at the cleavage site is also shown. Abbreviations: HDE, histone downstream element; SL, stem loop, (c) wtHSE time dependent in vitro cleavage assay using a 5' or an internally labeled [32P] unmodified (WT RNA) or a single nucleotide substituted (MUT RNA) histone pre-mRNA fragment (as in b). Extended Data Table 1.

Native HSE

Data collection

Space group PI

Cell dimensions

a, b, c (A) 62.95, 67.13, 67.90

α, β, γ{°) 109.31 , 105.40, 90.17

Resolution (A) 45.96 - 1.79 (1.84 - 1.79 A) a

R b 0.10 (0.81)

I/a(I) 12.4 (2.6)

Completeness (%) 95.5 (92.6)

Redundancy 6.9 (6.7)

Refinement

Resolution (A) 45.95 - 1.79

No. reflections 90641

R 0.182/0.21 1

No. atoms

Protein 6235

Ligand/ion 38

Water 514

B factors

Protein 27.27

Ligand/ion 44.40

Water 33.94

R.m.s. deviations

Bond lengths (A) 0.01

1.37

Extended Data Table 1. Crystallographic data and refinement statistics PDB ID 4V0H. a Values in parentheses are for highest-resolution shell.

Rmerge =∑/, \ hi - <IA> | /∑A∑i<h> where lu is the /th observation of reflection h, and <I¾> is the mean intensity of that reflection. c Rwork =∑ || F 0 bs |-|F C aic||/|F 0 b S | x 100. d Rfree is calculated in the same way as Rwork but using a test set containing 5.01 % of the data, which were excluded from the refinement calculation.

Extended Data Table 2

Extended data Table 2. Oligonucleotide primers and single guide RNA targeting sequences used in site directed mutagenesis, RT-qPCR experiments and CRISPR/CAS9 mediated KD. Abbreviations: GB, gene body; RT, read through. Note that the standard BBL numbering system has been used 25 . SEQUENCE INFORMATION SEQ ID NO: 1

HSE sp |A4D2B0 | MBLCl_HUMAN Metallo-beta-lactamase domain-containing protein 1 0S=Homo sapiens GN=MBLAC1 PE=1 SV=1

MRTEPLCGASPLLVPGDPYSWVLLQGYAEPEGVGDAVRADGSVTLVLPQTRGPASSH RE

SPRGSGGAEAALEEAARGPILVDTGGPWAREALLGALAGQGVAPGDVTLVVGTHGHS DHI

GNLGLFPGAALLVSHDFCLPGGRYLPHGLGEGQPLRLGPGLEVWATPGHGGQRDVSV WA

GTALGTVWAGDVFERDGDEDSWQALSEDPAAQERSR RVLVVADWVPGHGPPFRVLRE

ASQPETEGGGNSQQEPWGDEEPALH

SEQ ID NO: 2

HSE substrate. The A82 restriction site is shown in bold underline

AUGCCGUGGAGAGCGGGCUUAAGAAGUGGCGGUUCGGCCGGAGGUUCCAUCGUAUCC

AAAAGGCUCUUUUCAGAGCCACCCACAUCAGCACUUGGAAGAAGCUGUACCGCUUGC C

CUCCGUGCUCCUCCGGCAUUAGAGCGGGGAAGGCACUUCCGCUUAGGCAAGCU




 
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