Functional genomics identifies a requirement of pre-mrna splicing factors for sister chromatid cohesion

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1 The EMBO Journal Peer Review Process File - EMBO Manuscript EMBO Functional genomics identifies a requirement of pre-mrna splicing factors for sister chromatid cohesion Sriramkumar Sundaramoorthy, María Dolores Vázquez-Novelle, Sergey Lekomtsev, Michael Howell and Mark Petronczki Corresponding author: Mark Petronczki, Cancer Research UK London Research Institute Review timeline: Submission date: 14 February 2014 Editorial Decision: 01 April 2014 Revision received: 03 August 2014 Accepted: 12 August 2014 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) Editor: Anne Nielsen 1st Editorial Decision 01 April 2014 Thank you for submitting your manuscript for consideration by The EMBO Journal and my apologies for the unusual duration of the review period in this case. Your study has now been seen by three referees whose comments are shown below. As you will see from the reports, all referees express interest in the findings reported in your manuscript and emphasize the high technical quality of the experimental data. However, while ref #1 and ref #2 consequently favour publication following minor revisions, ref #3 is more critical and questions the overall novelty and specificity of findings. In light of these rather divergent recommendations, we had an additional round of consultation with all three refs to specifically address the conceptual concerns raised. The outcome of these discussions is that while splicing in general may be expected to impact on numerous biological pathways (as pointed out by ref #3), the main finding of your work is the surprisingly specific effect on sororin - as acknowledged by all three referees and strongly supported by refs #1 and #2 in these additional discussions. We would therefore be happy to pursue publication of an adequately revised version of your manuscript. We realize that some of the suggestions made by ref #3 may be outside the scope of the present work, but we would ask you to focus your efforts on the following points: -> Please include controls for knockdown efficiency (ref#1) -> Please comment on the general correlation between observed phenotypes and sororin expression EMBO 1

2 The EMBO Journal Peer Review Process File - EMBO levels (refs #1 and #2) -> Please provide further experimental data to address the mechanistic basis for the high sororin sensitivity to altered splicing (refs #2 and #3) by -> assessing mrna transcription and turnover rates for sororin and controls -> testing if splicing sensitivity is correlated with specific sequence features -> addressing splicing sensitivity for other cohesin factors -> In addition, please discuss/address all minor issues raised by all three referees Given the referees' positive recommendations, we offer you the opportunity to submit a revised version of the manuscript, addressing the comments of all three reviewers. I should add that it is EMBO Journal policy to allow only a single round of revision, and acceptance or rejection of your manuscript will therefore depend on the completeness of your responses in this revised version. Please do not hesitate to contact me if you have questions related to the review process and the requests made by the referees. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process, please visit our website: We generally allow three months as standard revision time. As a matter of policy, competing manuscripts published during this period will not negatively impact on our assessment of the conceptual advance presented by your study. However, we request that you contact the editor as soon as possible upon publication of any related work, to discuss how to proceed. Should you foresee a problem in meeting this three-month deadline, please let us know in advance and we may be able to grant an extension. Thank you for the opportunity to consider your work for publication. I look forward to your revision. REFEREE REPORTS: Referee #1: Based on RNAi screening, the authors found that the depletion of MFAP1 causes abnormal nuclear morphology, a hallmark of the defect in mitotic progression. They confirmed that mitotic progression is defective due to a cohesion defect in MFAP1-depleted cells. Other splicing factors such as SART1 also cause a similar phenotype. Moreover, the authors show that the cohesion defect is detected in interphase, and that the stability of the cohesin-chromatin interaction becomes less stable in MFAP1-depleted cells. They examined the expression of sororin, which maintains the stable association of cohesin with chromatin until mitosis. The sororin protein levels are decreased in MFAP1-depleted cells due to a defect in the splicing of the sororin pre-mrna. Cells expressing an intronless version of sororin rescue the cohesion defect seen in MFAP1-depleted cells. Furthermore, the depletion of Wapl, a counteracter of sororin, also rescues the phenotype. Thus, the authors conclude that splicing factors are required for the maintenance of cohesion and faithful mitotic progression, and that the splicing of the sororin mrna is the critical target for this pathway. Overall, the work in the paper is of high quality, and for the most part supports the important conclusions reported. Comments; 1) Depletion of splicing factors and Wapl should be examined by immunoblot. (Fig.3 and Fig. 8). 2) In general the phenotype caused by RNAi looks very strong (~100%). Please explain this measurement in detail. EMBO 2

3 The EMBO Journal Peer Review Process File - EMBO Referee #2: The manuscript by Sundaarmoorthy et al. describes the relevance of multiple pre-mrna splicing factors for sister chromatid cohesion. Although there were previous reported evidences for mitotic defects induced by defective splicing, the molecular reason for these observations was not clear. The manuscripts by Petronczki's and Peters' groups make a strong link between the splicing machinery and the protein levels of sororin as a consequence of aberrant splicing. The study by Sundaarmoorthy et al therefore addresses a very important issue and the results are exciting, novel and of high quality. In general, the manuscript is scientifically sound and technically very solid. The analyses are meticulously performed and the take-home message is very significant for our understanding on the links between gene transcription, splicing, chromosome segregation and, perhaps, cancer biology. Hence, I strongly recommend publication. I only have some minor comments that the authors may consider to improve the manuscript. How the phenotypes observed in Fig. 1B compare to downregulation of sororin? It seems from this panel that the cell fate after MFAP1 sirna is very diverse. Did cells die also in interphase? It is difficult to see single chromatids in Fig. 3B (/b etc.) and it will be even worst in the reduced published figure so adding insets may help to understand the phenotype. Some insets sin other panels (e.g. 4A) could also be enlarged. Statistics are missing in some histograms (Fig. 6). Why sororin? Is it any clue from the analysis about which specific sequences make the sororin gene susceptible to the lack of splicing factors? Again, these are minor points and do not diminish my enthusiasm for this study. Referee #3: In this study the authors aimed at identifying new factors involved in mitosis. Based on published datasets, the authors established a list of 718 potential candidates whose involvement in mitosis were further tested by a systematic sirna-based screen. From 718 candidates the list shrunk to 50 genes whose sirna appeared to have a real impact on nuclear morphology. This list was further reduced to 7 confirmed genes whose depletion had a consistent effect on nuclear shape. From this list, the depletion of the splicing factor MFAP1 resulted in particularly severe nuclear fragmentation. By using imaging techniques, the authors determined that in cells lacking MFAP1, chromosomes could not be aligned at the metaphase plate due to a loss of sister chromatid cohesion, suggesting that MFAP1 could have a function in sister chromatid cohesion in addition to its well characterized role as core pre-mrna splicing factor. However, by performing sirna-mediated depletions, the authors identified 18 other splicing factors that had a similar effect on chromatid cohesion as that observed upon MFAP1 depletion. Thus, the authors convincingly established a general link between pre-mrna splicing and chromatid cohesion, but apparently no specific or direct effect of MFAP1 on mitosis. Further experiments excluded a potential defect in cohesin loading onto chromatin, but demonstrated that loss of spliceosome components resulted in a specific and dramatic decrease in the essential chromatid cohesion factor sororin. Consistent with a requirement for pre-mrna splicing, complementation experiments in which sororin was expressed from an intronless transgene restored sister chromatid cohesion in cells depleted of splicing factors. This study demonstrates that the integrity of the spliceosome is required to maintain an appropriate level of spliced sororin mrna and its encoded protein sororin, which plays a role in chromatid cohesion. This study is well written and understandable to a large audience. The experimental procedures are well described and controlled. However, spliceosomal factors are essential for virtually all cellular processes, as splicing is an upstream event in gene expression and literally controls the expression of nearly all genes in mammalian cells. Therefore, the question addressed in this study does not represent a significant step forward in our understanding of the chromatid cohesion process, in addition to failing to identify new factors directly involved in mitosis. Thus, this study is not suitable for publication in EMBO J. in its present form. EMBO 3

4 The EMBO Journal Peer Review Process File - EMBO An interesting finding in this study is that the protein levels of the essential chromatid cohesion factors SMC1, SMC3, SCC1 and SA2 are not altered upon sirna depletion of the essential splicing factors NHP2L1, MFAP1, SART1 and CDC5L. Only the level of the sororin protein appears to be reduced. One obvious explanation would be that sororin has a high rate of transcription and protein turnover, which would explain its acute sensitivity to a reduction in splicing activity. However this point is not investigated and only briefly mentioned in the discussion. This manuscript would greatly benefit if further experiments providing insight into how stalling a general process like splicing appears to affect only one chromatid cohesion factor, i.e. sororin. Therefore I would greatly encourage the authors to determine sororin's half-life and its transcription rate, and determine whether this provides a satisfying explanation for sororin's exceptional sensitivity towards a general reduction in splicing. In addition the authors could compare the effect of depletion of spliceosome components on sororin to other known short lived proteins. Moreover, the authors should investigate whether only the splicing of sororin is blocked upon NHP2L1, MFAP1, SART1 and CDC5L depletion or whether the splicing of the other cohesion factors SMC1, SMC3, SCC1 and SA2 is also affected. In summary, any experiment that would help us understand the specific effect observed on sororin would greatly improve the significance and general interest of this manuscript. 2. Page 5: For 7 genes, two or more out of four sirna duplexes recapitulated the phenotype observed in the primary screen (Supplementary Table 2). This suggests that many of 50 hits selected from the primary screen were caused by off-target depletion effects. These are not necessarily off-target effects. A single RNAi duplex does not always inhibit efficiently gene expression. 3. Page 9: Like MFAP1, NHP2L1 and SART1 are components of the U5 and U4/U6 tri-snrnp complex MFAP1 is not a component of tri-snrnp but rather a non-snrnp, protein factor that joins the spliceosome at the spliceosomal B complex stage. 4. Page9:...Prp19 complex that promotes the catalytic activation of the spliceosome for cleavage at the 5' splicing site This should be rephrased to "...Prp19 complex required for the activation of the spliceosome before the first step of the splicing reaction" 5. Fig. 1B vs Fig. S1: could the authors explain the difference in penetrance observed. In Fig. 1B only 16 out of 50 cells successfully passed cell division while in Fig. S1 the defects were observed in only ~25% cells. 6. Fig. 4C: a loading control is missing 7. Fig. 6A: it seems that sirna-mediated depletion of sororin leads to co-depletion of SCC1. Does sororin affect the level of SCC1? Why don't CDC5L cells, which have a dramatically reduced level of sororin, show any reduction in SCC1? 8. Fig. 6C: in contrast to MFAP1 and NHPL2L1, SART1 and CDC5L cells show a two-fold reduction in the level of Exon2 of sororin (pre)-mrna. Is this a significant reproducible change? If so, do these cells have a transcription related defect (in the case of the sororin gene)? What is the steady state ratio of the sororin pre-mrna to its mrna? What is the half-life of sororin mrna? 9. Fig. 7 shows much milder effects of splicing factor knockdown as compared to Fig. 3 and Fig. S2, even in the case of CDC5L for which RNAi treatment was performed for nearly the same time. Could the authors comment on this. 10. Fig. 8B: the cartoon is confusing. "Premature loss of chromatid cohesion" should be next to free cohesion, right? Please indicate in the legend the subunits of the cohesin "triangles". 11. Page 12 (and Page 16 of Discussion): Expression of the intronless sororin transgene did not however restore mitotic progression in cells transfected with sirnas targeting spliceosome subunits... It is not clear based on which data this conclusion has been made. 12. Page 17: in the last paragraph of the Discussion the authors describe the potential biomedical implications of their findings, in particular to chronic lymphocytic leukaemia. Defects in the splicing factors described in this manuscript lead to mitotic arrest or cell death rather than uncontrolled cell proliferation characteristic for chronic lymphocytic leukaemia. Please clarify this point. 1st Revision - authors' response 03 August 2014 EMBO 4

5 Response to referees comments EMBOJ We would like to thank all reviewers for their comments, suggestions and criticism. To address the comments, we have added new experiments and Figures to the revised version of our manuscript and we have made changes to the manuscript text. Also we include an additional figure at the end of this document. Please find below a point-bypoint response to the reviewers comments. Referee #1: Based on RNAi screening, the authors found that the depletion of MFAP1 causes abnormal nuclear morphology, a hallmark of the defect in mitotic progression. They confirmed that mitotic progression is defective due to a cohesion defect in MFAP1- depleted cells. Other splicing factors such as SART1 also cause a similar phenotype. Moreover, the authors show that the cohesion defect is detected in interphase, and that the stability of the cohesin-chromatin interaction becomes less stable in MFAP1- depleted cells. They examined the expression of sororin, which maintains the stable association of cohesin with chromatin until mitosis. The sororin protein levels are decreased in MFAP1-depleted cells due to a defect in the splicing of the sororin premrna. Cells expressing an intronless version of sororin rescue the cohesion defect seen in MFAP1-depleted cells. Furthermore, the depletion of Wapl, a counteracter of sororin, also rescues the phenotype. Thus, the authors conclude that splicing factors are required for the maintenance of cohesion and faithful mitotic progression, and that the splicing of the sororin mrna is the critical target for this pathway. Overall, the work in the paper is of high quality, and for the most part supports the important conclusions reported. Comments; 1) Depletion of splicing factors and Wapl should be examined by immunoblot. (Fig.3 and Fig. 8). In the revised version of our manuscript we have added immunoblots probing the depletion of WAPL and that of splicing factors, against which antibodies were available. These new data are now included as Expanded View Figure E3A and E8B. Furthermore, the WAPL + splicing factor co-depletion experiment aimed at testing suppression of cohesion defects was repeated. Figure 8A now displays the mean values for cohesion status from the 2 repeats. 2) In general the phenotype caused by RNAi looks very strong (~100%). Please explain this measurement in detail. The depletion of several factors (incl. sororin, SGOL1, SCC1, CDC5L, ) causes highly penetrant defects in sister chromatid cohesion as detected by chromosome spread assays. In our experience, highly penetrant cell division phenotypes can be achieved by reverse transfection of sirna duplexes into human cells depending on the function and protein level threshold requirement of the particular factor targeted (e.g.: Lekomtsev et al., Naure 2012; Su et al., DevCell 2011). Chromosome spreading from mitotic cells is likely to exacerbate cohesion defects to a small degree as even in control cells around 10% of spread karyotypes can display a loss of cohesion. To address, this we conducted FISH experiments in intact mitotic cells following MFAP1 depletion (Fig. 1B). These

6 experiments demonstrated that centromeres of sister chromatids were separated in mitotic cells even without the procedure of chromosome spreading. In our manuscript we have followed the conventional classification of sister chromatid cohesion status into three categories of increasing phenotypic severity: normal (X-shaped), parallel chromatids without connection at central construction, and fully split (no alignment of chromatids observed). For each experiment or experimental repeat, 100 spread karyotypes (derived from 100 cells) were assessed (as detailed in Figure legends) and classified according to the aforementioned categories. Within the karyotype/chromosome spreads of a single cell, the vast majority of chromatids or chromosomes typically conforms to one of the categories and the spread was therefore classified accordingly. A value of e.g. 90% split sister chromatids in graphs is the result of 100 observed karyotypes displaying no association between sister chromatids. For percentages of split sister chromatids specified in micrographs, the categories of karyotypes with parallel and fully split chromatids were combined. We agree that this could be confusing and thank the reviewer for pointing this out. To clarify this point we have added a short description to the material and methods section of the revised manuscript. Also we have changed the wording for in the % associated with micrographs to % spreads with separated chromatids. Referee #2: The manuscript by Sundaarmoorthy et al. describes the relevance of multiple premrna splicing factors for sister chromatid cohesion. Although there were previous reported evidences for mitotic defects induced by defective splicing, the molecular reason for these observations was not clear. The manuscripts by Petronczki's and Peters' groups make a strong link between the splicing machinery and the protein levels of sororin as a consequence of aberrant splicing. The study by Sundaarmoorthy et al therefore addresses a very important issue and the results are exciting, novel and of high quality. In general, the manuscript is scientifically sound and technically very solid. The analyses are meticulously performed and the take-home message is very significant for our understanding on the links between gene transcription, splicing, chromosome segregation and, perhaps, cancer biology. Hence, I strongly recommend publication. I only have some minor comments that the authors may consider to improve the manuscript. How the phenotypes observed in Fig. 1B compare to downregulation of sororin? It seems from this panel that the cell fate after MFAP1 sirna is very diverse. Did cells die also in interphase? To compare the effect of loss of sororin to the effect of loss of MFAP1, we have analyzed the fate of cells and their mitotic phenotype by live-cell imaging following transfection of sororin sirna. The cell fate data and mean mitotic duration following sororin depletion are presented in a Figure for Referees that has been attached at the end of this letter. As shown in the Figure, loss of sororin causes a fully penetrant mitotic arrest with 70% of cells undergoing cell death during the arrest and the remaining cells exiting mitosis in an aberrant fashion. The mean time of sororin-depleted cells spent in mitosis was 678 min compared to 510 min for loss of MFAP1 (control 46 min) (Fig. 1B). Thus, the loss of sororin leads to a slightly more uniform and penetrant mitotic defect than

7 MFAP1 loss. The effect of MFAP1 loss is more pleiotropic due to the protein s role in splicing. Nevertheless, loss of MFAP1 (and other splicing factors) causes a mitotic phenotype consistent with a reduction in sororin levels, with the majority of cells dying during a mitotic arrest or exiting mitosis in an aberrant fashion following a protracted arrest. The terminal phenotype of loss of MFAP1 (and other splicing factors) is cell death. This is presumably caused by combination of defective mitosis and gene expression failure. A minor fraction (10%) of MFAP1-depleted cells died prior to entry into mitosis. For mitotic duration analysis only cells entering mitosis were scored. In summary, our data suggest that loss of MFAP1 causes a more pleiotropic defect than depletion of sororin finally resulting in cell death. However, prior to cell death the vast majority of MFAP1-depleted cells arrest in mitosis with split sister chromatids in a mitotic checkpoint-dependent manner (Figure 1). It is difficult to see single chromatids in Fig. 3B (/b etc.) and it will be even worst in the reduced published figure so adding insets may help to understand the phenotype. Some insets sin other panels (e.g. 4A) could also be enlarged. We thank the reviewer for pointing this out. In the revised version of the manuscript we have included enlarged insets of chromosomes/chromatids for Figure panels 2A, 2C, 3B and 8A. Furthermore, we have enlarged the insets of FISH signals shown in Figure 4A. Statistics are missing in some histograms (Fig. 6). For the revised version of the manuscript, we have repeated the immunoblot analysis shown in Fig. 6A and we have conducted a new qrt-pcr analysis (three biological repeats) of intron retention for sororin and core cohesin subunit RNAs following depletion of splicing factors. Figure 6 now shows the mean of the data obtained from the biological repeats and contains error for standard deviation. Why sororin? Is it any clue from the analysis about which specific sequences make the sororin gene susceptible to the lack of splicing factors? We have explored this interesting question. Analysis of 5 and 3 splice site sequences (MaxEntScan splice site score algorithm; and the presumptive position of the branch site with regard to the 3 splice site in of the first intron of sororin did not reveal particular features that would explain a pronounced sensitivity of sororin pre-mrna splicing to the depletion of spliceosomes. In the future, global analysis of spliceosome assembly and branch site usage may be able to address this question if correlated with aberrant intron retention upon loss of specific spliceosome components. Based on the transcriptome-wide analysis of intron retention in the accompanying manuscript by the Peters lab, it is clear that depletion of the splicing factor SNW1 affects intron removal in a number of genes thus suggesting that this effect is not unique to sororin. However, amongst the factors essential for cohesion, depletion of spliceosome subunits had the strongest impact on intron retention in sororin RNA (Fig. 6B). In the revised version of the manuscript we show that both sororin protein and mrna have a higher turnover than other cohesin subunit (Fig 6C and E7). In G1 cells sororin protein has an extraordinary short half-life of less than 2 hours (Fig. 6C and E7B), consistent with it having been identified as an APC/C substrate (Rankin et al., 2005). This short protein half-life sets sororin apart from other cohesin proteins. The high turnover of

8 sororin RNA and protein gene products may synergize with a potentially stringent requirement for intact spliceosomes for intron removal to cause a drastic and selective reduction of sororin protein following depletion of splicing factors. We have amended the discussion section of our revised manuscript to include the new data and to better reflect the aforementioned points. Again, these are minor points and do not diminish my enthusiasm for this study. Referee #3: In this study the authors aimed at identifying new factors involved in mitosis. Based on published datasets, the authors established a list of 718 potential candidates whose involvement in mitosis were further tested by a systematic sirna-based screen. From 718 candidates the list shrunk to 50 genes whose sirna appeared to have a real impact on nuclear morphology. This list was further reduced to 7 confirmed genes whose depletion had a consistent effect on nuclear shape. From this list, the depletion of the splicing factor MFAP1 resulted in particularly severe nuclear fragmentation. By using imaging techniques, the authors determined that in cells lacking MFAP1, chromosomes could not be aligned at the metaphase plate due to a loss of sister chromatid cohesion, suggesting that MFAP1 could have a function in sister chromatid cohesion in addition to its well characterized role as core pre-mrna splicing factor. However, by performing sirna-mediated depletions, the authors identified 18 other splicing factors that had a similar effect on chromatid cohesion as that observed upon MFAP1 depletion. Thus, the authors convincingly established a general link between pre-mrna splicing and chromatid cohesion, but apparently no specific or direct effect of MFAP1 on mitosis. Further experiments excluded a potential defect in cohesin loading onto chromatin, but demonstrated that loss of spliceosome components resulted in a specific and dramatic decrease in the essential chromatid cohesion factor sororin. Consistent with a requirement for pre-mrna splicing, complementation experiments in which sororin was expressed from an intronless transgene restored sister chromatid cohesion in cells depleted of splicing factors. This study demonstrates that the integrity of the spliceosome is required to maintain an appropriate level of spliced sororin mrna and its encoded protein sororin, which plays a role in chromatid cohesion. This study is well written and understandable to a large audience. The experimental procedures are well described and controlled. However, spliceosomal factors are essential for virtually all cellular processes, as splicing is an upstream event in gene expression and literally controls the expression of nearly all genes in mammalian cells. Therefore, the question addressed in this study does not represent a significant step forward in our understanding of the chromatid cohesion process, in addition to failing to identify new factors directly involved in mitosis. Thus, this study is not suitable for publication in EMBO J. in its present form. We thank the reviewer for his/her comments and criticism. Our work demonstrates that one of the acute consequences of compromised splicing in human cells is a failure of cohesin to stably associate with chromatin and to hold sister chromatids together. Furthermore, we provide the mechanistic basis for this effect. Cohesin and sister chromatid cohesion are essential for several pivotal chromosomal processes including chromosome segregation, DNA damage response and repair, and gene regulation. Our work highlights an early cellular consequence of derailing a major gene expression system in human cells. Furthermore, it emphasizes the delicate balance that cells have to strike between the WAPL-dependent cohesin release activity and the sororin-

9 dependent mechanism that counteracts WAPL and promotes the establishment of long-lived connections between sister chromatids. It also provides one explanation for the reported involvement of spliceosome subunits in mitosis. One of the splicing factors that we identify to be important for sister chromatid cohesion, SF3B1, has recently emerged as one of the major recurrently mutated driver genes in hyperproliferative haematopoietic disorders, such as chronic lymphocytic leukaemia (CLL) and myelodysplastic syndrome (MDS). Heterozygous somatic mutations in SF3B1 are detected in CLL cells as frequently as mutations in the classic tumor suppressor p53 (Wang et al., NEJM 2011; Rossi et al., Blood 2011; Queseda et al., Nature Genetics 2011; Landau et al., Cell 2013). SF3B1 mutations occur in 65% of patients suffering from a subtype of MDS (MDS with ring sideroblasts) (PMIDs: Papaemmanuil et al., NEJM 2011; Yoshida et al., Nature 2011). Our findings connecting SF3B1 to sister chromatid cohesion and cohesin s association with chromatin may therefore have clinical implications and could inform about aspects of CLL and MDS pathology. Testing and modelling the effects of patient mutations in SF3B1 on cohesin dynamics is a goal worth pursuing in the future. Based on the aforementioned points, we believe that reporting on the connection between splicing and cohesin dynamics/cohesion that we have uncovered here is important and relevant. 1. An interesting finding in this study is that the protein levels of the essential chromatid cohesion factors SMC1, SMC3, SCC1 and SA2 are not altered upon sirna depletion of the essential splicing factors NHP2L1, MFAP1, SART1 and CDC5L. Only the level of the sororin protein appears to be reduced. One obvious explanation would be that sororin has a high rate of transcription and protein turnover, which would explain its acute sensitivity to a reduction in splicing activity. However this point is not investigated and only briefly mentioned in the discussion. This manuscript would greatly benefit if further experiments providing insight into how stalling a general process like splicing appears to affect only one chromatid cohesion factor, i.e. sororin. Therefore I would greatly encourage the authors to determine sororin's half-life and its transcription rate, and determine whether this provides a satisfying explanation for sororin's exceptional sensitivity towards a general reduction in splicing. In addition the authors could compare the effect of depletion of spliceosome components on sororin to other known short-lived proteins. Moreover, the authors should investigate whether only the splicing of sororin is blocked upon NHP2L1, MFAP1, SART1 and CDC5L depletion or whether the splicing of the other cohesion factors SMC1, SMC3, SCC1 and SA2 is also affected. In summary, any experiment that would help us understand the specific effect observed on sororin would greatly improve the significance and general interest of this manuscript. We have addressed several of these points and included new experiments and dataset in the revised version of our manuscript. Analysis of 5 and 3 splice site sequences (MaxEntScan splice site score algorithm; and the position of the presumptive branch site (TAACT motif present) with regard to the 3 splice site in of the first intron of sororin did not reveal particular features that would explain a pronounced sensitivity of sororin pre-mrna splicing to the depletion of spliceosomes. Based on the transcriptome-wide analysis of intron retention in the accompanying manuscript by the Peters lab, it is clear that depletion of the splicing factor SNW1 affects intron removal in a number of genes. This suggests that the effect is not unique to sororin. However, amongst the factors essential for cohesion, depletion of spliceosome subunits had the strongest impact on intron retention in sororin RNA (Fig. 6B). In a new set of experiments (triplicate biological repeat), we used intron-exon junction specific

10 primer pairs to track intron retention. We now demonstrate that enhanced intron retention in sororin RNA but not in RNAs encoding the core cohesin subunits SCC1, SA2, SMC3 or SMC1 (Fig. 6B). Thus, our results support a selective effect on sororin splicing. While the molecular basis for this selectivity remains to be determined, we found that both sororin protein and mrna have a higher turnover than other cohesin subunit in the revised version of the manuscript (Fig 6C and E7). For these experiments, cells were treated with cycloheximide or actinomycin D. In G1 cells sororin protein has an extraordinarily short half-life of less than 2 hours (Fig. 6C and E7B). This short protein half-life sets sororin apart from other cohesin proteins. Our targeted analysis is supported by a yet unpublished global analysis of protein and RNA half-life by the Selbach lab (Berlin) that utilizes metabolic pulse labeling (as developed in Schwannhäuser et al., Nature 2011) (Henrik Zauber and Matthias Selbach, personal communication). The high turnover of sororin RNA and in particular sororin protein may synergize with a potentially stringent requirement for intact spliceosomes for intron removal to cause a drastic and selective reduction of sororin protein following depletion of splicing factors. We have amended the discussion section of our revised manuscript to include the new data and to better reflect the aforementioned points. In summary, our newly included data indicate the selective retention of introns in sororin RNA following splicing factor depletion compared to the RNAs of other cohesin subunits. Furthermore, we provide data demonstrating higher turnover of sororin gene products compared to the products of core cohesin subunits. The mechanistic basis for a selective or stringent requirement of intact spliceosomes for the splicing of sororin RNA (and others) remains unknown at this point. It will be facilitated by the global analyses of spliceosome assembly on RNA and branch site usage if correlated with aberrant intron retention upon loss of specific spliceosome components. In our opinion, deciphering why sororin RNA splicing is particularly sensitive to the depletion of spliceosome components, although clearly an interesting question, is beyond the scope of this manuscript. 2. Page 5: For 7 genes, two or more out of four sirna duplexes recapitulated the phenotype observed in the primary screen (Supplementary Table 2). This suggests that many of 50 hits selected from the primary screen were caused by off-target depletion effects. These are not necessarily off-target effects. A single RNAi duplex does not always inhibit efficiently gene expression. In the revised version of the manuscript, we have removed the sentence suggesting that the remaining 43 primary screen hits were caused by off-target effects. 3. Page 9: Like MFAP1, NHP2L1 and SART1 are components of the U5 and U4/U6 trisnrnp complex MFAP1 is not a component of tri-snrnp but rather a non-snrnp, protein factor that joins the spliceosome at the spliceosomal B complex stage. We thank the reviewer for pointing out this mistake. We have removed Like MFAP1, from the above sentence. 4. Page9:...Prp19 complex that promotes the catalytic activation of the spliceosome for cleavage at the 5' splicing site This should be rephrased to "...Prp19 complex required for the activation of the spliceosome before the first step of the splicing reaction" The sentence has been rephrased based on the reviewer s suggestion.

11 5. Fig. 1B vs Fig. S1: could the authors explain the difference in penetrance observed. In Fig. 1B only 16 out of 50 cells successfully passed cell division while in Fig. S1 the defects were observed in only ~25% cells. The apparent discrepancy in phenotypic penetrance between Fig. 1B and Fig S1 (now E1) stems from the different methods of analysis. For Figure 1B, the fate of cells was tracked over a long period using time-lapse microscopy (from 32 hrs post transfection onwards). For Figure S1/E1, the analysis was performed by fixing cells at a particular timepoint following sirna transfection (at 52 hrs post transfection). The latter approach presents a terminal and static picture of cellular phenotypes. Many MFAP1 depleted cells underwent mitotic arrest and cell death (in mitosis) before fixation. These cells will be lost and not captured using a fixed cell analysis by immunofluorescence. However, time-lapse tracking allows us to analyze and score the fate of all cells over time. Thus, the phenotypic penetrance is lower in Figure S1/E1 as strongly depleted cells undergo mitotic arrest/cell death (as shown in Fig. 1B) before the time point of analysis. In our experience, time-lapse recording present a more reliable view of the mitotic aberrations and phenotypic penetrance. 6. Fig. 4C: a loading control is missing To provide a loading control, we have probed the samples with an antibody against α-tubulin (see Figure 4C). Please note that the level of SMC3 acetylation was calculated using the ratios of AcSMC3 signal intensity to SMC3 signal intensity for each condition (using detection of fluorescently labeled secondary antibodies). 7. Fig. 6A: it seems that sirna-mediated depletion of sororin leads to co-depletion of SCC1. Does sororin affect the level of SCC1? Why don't ΔCDC5L cells, which have a dramatically reduced level of sororin, show any reduction in SCC1? We have conducted the quantitative immunoblot analysis of sororin protein levels shown in Figure 6A twice. In both experiments, sororin sirna transfection caused a reduction in SCC1 protein levels to around 50%. The analysis was performed in G2 synchronized cells. Two possible interpretations could account for this effect. (1) complete depletion of sororin interferes with the stability of SCC1 protein by e.g. increasing the degradation of SCC1 in the soluble pool. (2) sororin sirna could affect SCC1 expression through off-target effects. If the latter is correct, the reduction of SCC1 does not measurably contribute to cohesion defects in sororin sirna transfected cells as of expression of an sirna-resistant sororin transgene completely restores cohesion after depletion of the endogenous counterpart (Fig. 7A). If the former hypothesis is correct, strong but incomplete reduction in sororin through depletion of splicing factors is not sufficient to destabilize SCC1. At this point, the basis of this reduction in SCC1 levels following sororin depletion is unclear. However, in our opinion, this does not affect the main conclusions of our work. 8. Fig. 6C: in contrast to ΔMFAP1 and ΔNHPL2L1,ΔSART1 and ΔCDC5L cells show a two-fold reduction in the level of Exon2 of sororin (pre)-mrna. Is this a significant reproducible change? If so, do these cells have a transcription related defect (in the case of the sororin gene)? What is the steady state ratio of the sororin pre-mrna to its mrna? What is the half-life of sororin mrna?

12 In the revised version of our manuscript we have replaced the intron retention analysis with a new set of data (Fig. 6B). In this analysis we detected selective strong retention (6 to 16 fold) of sororin introns 1 and 2 following depletion of all tested splicing factors. In contrast, we do only detect minor changes in the retention of tested introns for SCC1, SA2, SMC3, and SMC1. The analysis used an exon only amplicon for normalization of intron-exon levels for all genes. This revealed that depletion of splicing factors reduced the combined levels of unprocessed + processed RNAs of all genes tested (sororin, SCC1, SA2, SMC3, and SMC1) around 1.5 to 3 fold depending on the splicing factor depleted. This suggests that interfering with splicing decreases the cellular transcript levels of the genes tested. However, sororin transcript levels were not reduced stronger than the levels of other genes tested. Only sororin RNA showed a strong increase in intron retention after normalization of intron-exon amplicon to the exon only amplicon for each gene (Fig 6B). 9. Fig. 7 shows much milder effects of splicing factor knockdown as compared to Fig. 3 and Fig. S2, even in the case of CDC5L for which RNAi treatment was performed for nearly the same time. Could the authors comment on this. In the case of CDC5L, the difference in phenotypic severity between Fig. 3 and Fig. 7 is explained by the fact that pools of 4 different sirna duplexes were used for depletion experiment in Figure 3 whereas a single sirna duplex (oligo 4) was used for the experiment in Fig. 7. As shown in the deconvolution dataset in Figure S2 (now E2), the CDC5L oligo4 caused a cohesion defect in 70% of spreads, which is slightly less than the 81% defect shown in Figure 7. Thus, for CDC5L, the phenotype obtained in Fig.7 is not milder than the one obtained in Fig. S2/E2 but actually slightly stronger. The use and identity of single sirna duplexes versus sirna pools duplexes is specified in the method section of the manuscript. The differences in phenotypic severity between Fig.3 and Fig. 7 for the other splicing factors (MFAP1, NHP2L1 and SART1) can be explained by the fact that cells were collected for chromosome spread analysis earlier than for the experiments in Fig. 3 and S2/E2. For Fig. 3 and S2/E2, cells were collected 56 hr post transfection (including 4 hr nocodazole treatment). In contrast, for the experiment in Fig 7 cells were collected after 40/34/40 hr for depletion of MFAP1/NHP2L1/SART1, respectively (all time intervals include 4 hr of nocodazole treatment). The timings are specified in the figure legends of the paper. The reason for a reduced exposure to sirnas for Fig7 was to reduce the impact of cohesion fatigue which can make a contribution to the loss of sister chromatid cohesion as mentioned in the discussion section of our manuscript and as elaborated on in more detail in the accompanying manuscript by the Peters lab. 10. Fig. 8B: the cartoon is confusing. "Premature loss of chromatid cohesion" should be next to free cohesion, right? Please indicate in the legend the subunits of the cohesin "triangles". We agree that the position of the text in the figure was not appropriate. In the revised version we have changed the position of the text box accordingly and placed the text box between the two separating chromatids. Furthermore, we have labeled the subunits of the cohesin complex in the model. 11. Page 12 (and Page 16 of Discussion): Expression of the intronless sororin transgene did not however restore mitotic progression in cells transfected with sirnas targeting spliceosome subunits... It is not clear based on which data this conclusion has been made.

13 To address this point, we have used time-lapse microscopy to measure the mitotic duration of cells expressing either an AcFL tag or an intronless and RNAi-resistant AcFL-tagged version of sororin (Figure E8B). Both stable transgenic cell lines were transfected with control sirna duplexes or duplexes targeting Sororin, MFAP1, NHP2L1, SART1 or CDC5L. Expression of the transgenic and sirna-resistant copy of sororin rescued the mitotic arrest following sororin sirna transfection but not following transfection with sirna duplexes targeting splicing factors. These data are now presented in Figure E8A of the revised manuscript and support the above statement in the manuscript. 12. Page 17: in the last paragraph of the Discussion the authors describe the potential biomedical implications of their findings, in particular to chronic lymphocytic leukaemia. Defects in the splicing factors described in this manuscript lead to mitotic arrest or cell death rather than uncontrolled cell proliferation characteristic for chronic lymphocytic leukaemia. Please clarify this point. Complete loss of splicing function as well as a penetrant defect in sister chromatid cohesion is likely to be cell lethal. This condition would clearly be incompatible with neoplastic growth in hyperproliferative disorders such CLL and MDS. However, the genetic changes detected in CLL patients are heterozygous somatic mutations. The most frequent mutation detected in SF3B1 in CLL patients is the somatic heterozygous mutation K700E. Our finding that depletion of SF3B1 results in dramatic loss of sister chromatid cohesion (Fig. 3A and B) raises the possibility that the heterozygous patient mutations such as K700E could alter the dynamics of cohesin-chromatin association and thereby change the fidelity of chromosome disjunction, DNA repair and gene regulation, all hallmarks of cancer, in a manner that is compatible with cell proliferation. Testing this hypothesis will require modeling the patient mutation K700E in a heterozygous and homozygous configuration in experimentally accessible cell models. This is an important goal for future research. Therefore, studies based on the findings presented in this manuscript could contribute to our understanding of the molecular basis of CLL and MDS. Given the prevalence of SF3B1 mutations in CLL and MDS, deciphering the impact of these mutations on cell physiology is of significant biomedical importance. To clarify the fact that disease-associated mutations of SF3B1 are not expected to be complete loss of-function situations we have added the term heterozygous to the discussion section of the revised version of our manuscript.

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15 The EMBO Journal Peer Review Process File - EMBO nd Editorial Decision 12 August 2014 Thank you for submitting your revised manuscript to The EMBO Journal. Your study has now been seen by two of the original referees (comments included below) and as you will see they both find that all criticisms have been adequately addressed. I am therefore happy to inform you that your manuscript has been accepted for publication with us. REFEREE REPORTS: Referee #1: I think that the authors properly addressed my concerns. The manuscript should be published immediately. Referee #2: The authors have properly addressed all my questions. In particular, I appreciate the effort to explain the specific requirement for the spliceosome machinery in maintaining sororin levels given the high turnover of this protein. I therefore recommend publication. EMBO 5