Splicing Factor HnRNP H Drives an Oncogenic Splicing Switch in Gliomas

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1 Manuscript EMBO Splicing Factor HnRNP H Drives an Oncogenic Splicing Switch in Gliomas Clare LeFave, Massimo Squatrito, Sandra Vorlova, Gina Rocco, Cameron Brennan, Eric Holland, Ying-Xian Pan, Luca Cartegni Corresponding author: Luca Cartegni, Memorial Sloan Kettering Cancer Center Review timeline: Submission date: 24 March 2011 Editorial Decision: 13 April 2011 Revision received: 28 June 2011 Accepted: 05 July 2011 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.) 1st Editorial Decision 13 April 2011 Thank you very much for submitting your research manuscript for consideration to The EMBO Journal editorial office. I did receive comments from three scientists that you will find enclosed for your information. The referees are in general positive about your results on hnrnp H in two distinct splicing events. However, additional controls and clarifications have been demanded. More importantly, ref#2 and #3 request experiments that are aimed at corroborating your findings in glioma cell lines and better correlate hnrnp H upregulation as potential causal event for the oncogenic phenotype. Overall, I am happy to offer you the chance to thoroughly amend the manuscript following the referee's requests. I also have to remind you that it is EMBO_J policy to allow a single round of major revisions and that the final decision on your manuscript entirely depends on content and strength of such an ultimate version. Yours sincerely, Senior Editor The EMBO Journal REFEREE REPORTS: Referee #1: European Molecular Biology Organization 1

2 LeFave et al. declare in their title that hnrnp H drives an oncogenic splicing switch in gliomas. This is supported by experiments showing that the splicing of gene IG20 exon 16 is inhibited by a silencer that appears to respond to hnrnp H, and that hnrnp H is expressed at elevated levels in gliomas. the work was extended to the proto-oncogene Ron, where the authors showed that each mrnp H had a significant effect on the inclusion of exon 11. Significantly, reducing the levels of hnrnp H decreased the ability of cells to migrate, but this activity was restored if the skipping of Ron exon 11 was enforced by other means. While restoration was incomplete and not wholly convincing, the result at least suggested that hnrnp H acts via Ron to increase cell migration. It is a shame that the authors did not go further and use mouse assays to show that hnrnp H could act as an oncogene. In general, the experiments were thorough and the results convincing. I have only two further observations to make: (1) Figure 3D shows a disappointing level of response of IG20 splicing to depletion of hnrnp H. Indeed, the increase from lanes 1 to 5 in the level of the inclusion may be matched by increases in the level of the skipped product. The RT-PCR results need to be quantified properly. The same point is made in Figure 3F and in the supplementary figures, so it might be better simply to remove 3D. (2) There is no evidence, as far as I can see, to show that the putative silencer in exon 16 mediates the effects of hnrnp H. It would be desirable to show that the mutations prevent any response to increased levels of hnrnp H. A minor point is that the top of page 10 refers to a Figure 1F. This does not exist. Indeed, there is no splicing redirection assay for IG20. Referee #2: In this interesting manuscript, the Cartegni lab describes a role for the RNA-binding protein hnrnp H in the regulation of alternative splicing of pre-mrnas associated with cancer (gliomas). There are several interesting aspects to this manuscript. First, the data clearly support a role for hnrnph in the regulation of alternative splicing leading to a pro-survival isoform of the death domain adaptor protein IG20-MADD and to the production of motility-enhancing isoform of the RON receptor tyrosine kinase. Importantly, the authors mapped the elements (ESS) responsible for the exon skipping event in both IG20 and RON. Furthermore, the authors also clearly show that hnrnp H promotes cell survival in HeLa cells and this effect is clearly mediated by inhibiting inclusion of IG20 exon 16. A particularly nice experiment is the one showing that the increased apoptosis following hnrnp H ablation can be rescued by targeted down-regulation of IG20 exon 16 (Fig. 3F). Along the same lines, LeFave and colleagues show that hnrnp H levels contribute to the invading properties in HeLa and T98G cells, and that this is at least in part due to hnrnp H- dependent modulation of RON exon 11 splicing. Overall, my impression of this work is very positive. It nicely links a thorough characterization of cis-acting sequences and trans-acting factors required for a regulated splicing event with the development of gliomas. This work would benefit from the following revisions Revisions - In the Discussion section, the authors claim that they should test the role of hnrnp H in tumor progression in-vivo using mouse models. This is clearly beyond the scope/time-frame of this manuscript. The authors should 1- test that hnrnp H blocks cell death via MADD activity in a glioma cell line (T98G cells) as they do in HeLa (figure 3F). 2- It is shown that hnrnp H is overexpressed in gliomas as the average of several samples but the European Molecular Biology Organization 2

3 variation between samples is quite high. The authors should analyze hnrnp H levels and IG20 exon 16- lacking variants (and/or RON exon 11) levels for some individual GBM samples to demonstrate that a clear correlation exists in Glioma. Minor comments - On page 4, when referring to SF2/ASF, the author should use the new nomenclature (SRSF1). Also, it would be good to properly introduce what SR proteins are. There are several updated review articles that can be used for this. - Authors seem to be missing from the first reference "Comprehensive genomic" Referee #3: In this work, LeFave et al. analyze the role of hnrnp H in regulating two important splicing events that occur in human gliomas and involve the IG20 and Ron genes. Overall, the work is well written and the results clear. Moreover, this work makes several points of interest regarding the multiple roles that might be played by a single splicing factor in tumor progression. 1) Understandably, most of the work is focused at elucidating the role played by hnrnp H in exon 16 of IG20 and exon 11 of Ron. However, this needs to be better addressed if the authors wish to make a clearer connection with disease. For example, on page 6 the authors mention the existence of several ESE elements within IG20 exon 16. Considering the low rate of exon inclusion it is probable that they play a major role in deciding exon 16 inclusion levels. However, aside from a preliminary bioinformatics analysis that might suggest the presence of SF2, SRp40, and SRp55 binding sites (Fig.2E) no confirmation has been made at the experimental level. Is overexpression of these factors capable of substantially improving exon 11 inclusion?. Such experiments might be a worthy addition as the authors may wish to use this information to measure the amount of these proteins in their glioma samples versus controls to make sure that the drop in exon exclusion may not be also ascribed to a drop in their expression (and not just to overexpression of hnrnp H). 2) the authors were unable to detect mron expression in the mouse samples of Fig.1E. But what about the cell lines used in 1D?. If detectable, do the trend in exon 11 inclusion/skipping in Ron mimic those of exon 16 in the IG20 gene?. If so this would be an important parallelism to further support the author's hypothesis. 3) In Fig.4F, the authors claim that multiple mutations in the two GGG motifs of Ron exon 11 enhance the switch towards exon inclusion. However, observing the wide variety of signals for the unspliced mrna at the top of the gel in the various lanes it is difficult to say if this is really true. Has this experiment been quantified in some way?. Moreover, the authors should check again the sequences provided in the 4E inset because the Ensembl entry (ENST ) for the human Ron gene shows the presence of a final G (..TGGGCG) at the end of the sequence they provide as opposed to the one they show (..TGGGCC). Finally, what happens to exon 11 inclusion levels when hnrnp H is silenced following this minigene transfection?. 3) In Ghigna et al., 2005, the silencer element that they identified in Ron exon 12 corresponds to nt of this exon that also contains at least one UGGG motif and several GGG repeats. Can these sequences bind hnrnp H too?, and if so, how did the authors rule out that the effect they see in Fig.4D may not be ascribed to this silencer sequence in exon 12?. Minor points: 1) the authors suggest that hnrnp F may not play a role in regulation because its levels are unaffected by hnrnp H depletion which can by itself rescue exon 16 skipping (page 8). Nonetheless, it would be interestingto know if hnrnp F is part of the complex assembled on the TGGG elements. Have the authors tried a supershift experiment with a hnrnp F specific antibody like thay have performed for hnrnp H?. European Molecular Biology Organization 3

4 1st Revision - authors' response 28 June 2011 Please find enclosed a revised copy of our manuscript "Splicing Factor HnRNPH Drives an Oncogenic Splicing Switch in Gliomas" (EMBOJ ). We believe we have convincingly addressed all the points raised by the reviewers and we have significantly re-written the manuscript to comply with EMBO length requirements (the main text is now 54,239 characters long, excluding the references). The text is now more concise and reads better. Some of the figures have been reorganized, with the introduction of significant new data. In particular : 1. The deletion analysis of IG20/MADD exon 16 where we mapped the putative ESEs was moved to the support information section (Supp Fig 2A-C), as suggested, to strengthen the focus on the hnrnph silencer. 2. We show that over-expression of splicing factors SRSF1, SRSF5 and SRSF6 has no effect on IG20/MADD splicing (Suppl. Fig 2D). 3. WE show that a single-point mutation of the ESS that causes exon 16 inclusion also voids the effect of hnrnph knock-down on IG20/MADD splicing (Figure 3E). 4. Co-transfection of hnrnph stimulates IG20/MADD intron exclusion. Again this does not happen when the ESS is mutated (Supp Fig s5) 5. We now show knocking-down hnrnph leads to cell death through splicing of ig20/madd exon 16 also in U373 glioma cells (in addition to HeLa cells). This data were moved from being described in a panel of figure 3 to heir own figure 4 (therefore there are now 7 figures). 6. RON splicing pattern was analyzed in normal tissues and in cell lines. 7. The ESS in exon 11 is necessary for hnrnph activity (Fig 5F). 8. Co-transfection of hnrnph stimulates RON exon 11 skipping. Again this does not happen when the ESS is mutated (Supp Fig s7b). 9. We analyzed the role of sequences in exon 12 of RON in exon 11 splicing, as suggested (Supp Fig s8). 10. We compare expression of hnrnph and splicing of IG20/MADD in individual GBM tumor samples and non-tumor samples (Supp Fig s12). 11. We show that BE(2) brain tumor cells induced to differentiate by Retinoic Acid express lower amounts of hnrnph, and at the same time show higher levels of IG20/MADD exon 16 (Supp Fig s13), supporting the notion that activation of hnrnph might reflect a stem-like state and developmental programs (as now more thoroughly addressed in the discussion) in addition some data previously indicted as not showní is now shown as supporting information (e.g.: Supp Fig s4). The discussion was significantly modified to shorten it and to reflect the new data. Here is a point-by point response to the refereeís comments: Referee #1: LeFave et al. declare in their title that hnrnp H drives an oncogenic splicing switch in gliomas. This is supported by experiments showing that the splicing of gene IG20 exon 16 is inhibited by a silencer that appears to respond to hnrnp H, and that hnrnp H is expressed at elevated levels in gliomas. the work was extended to the proto-oncogene Ron, where the authors showed that each mrnp H had a significant effect on the inclusion of exon 11. Significantly, reducing the levels of hnrnp H decreased the ability of cells to migrate, but this activity was restored if the skipping of Ron exon 11 was enforced by other means. While restoration was incomplete and not wholly convincing, the result at least suggested that hnrnp H acts via Ron to increase cell migration. It is a shame that the authors did not go further and use mouse assays to show that hnrnp H could act as an oncogene. European Molecular Biology Organization 4

5 In general, the experiments were thorough and the results convincing. I have only two further observations to make: (1) Figure 3D shows a disappointing level of response of IG20 splicing to depletion of hnrnp H. Indeed, the increase from lanes 1 to 5 in the level of the inclusion may be matched by increases in the level of the skipped product. The RT-PCR results need to be quantified properly. The same point is made in Figure 3F and in the supplementary figures, so it might be better simply to remove 3D. As the reviewer points out, increase in ig20/madd exon 16 inclusion following hnrnph depletion is shown in multiple other experiments and thus this panel is a bit redundant. However, we think it is important to highlight this effect in the Figure, so we substituted this figures with two separate ones that show also additional informations: 1) Figure 3E now shows the effect of hnrnph depletion on splicing of exogenous IG20/MADD exon 16, when it is expressed from the IG20/MADD minigene. In addition, the figure also shows that the increase in inclusion is dependent on the integrity of the ESS in exon 16, as a single point mutation abrogates the effect due to hnrnph knockdown (of course the baseline of inclusion is much higher with the mutant to begin with). This point also in part addresses point 2 below, as it provides evidence that the effect of hnrnph is mediated by the ESS 2) Figure 3F now shows the effect of hnrnph depletion on splicing of endogenous IG20/MADD exon 16, using two independent sirnas, which argues against off-site effects. (2) There is no evidence, as far as I can see, to show that the putative silencer in exon 16 mediates the effects of hnrnp H. It would be desirable to show that the mutations prevent any response to increased levels of hnrnp H. This is a good experiment. We have tried to ever-express hnrnph many times using different sources and backbone plasmids, but we were never able to observe a clear up-regulation of hnrnph in cells. This is possibly due to the high basal level of hnrnph expression in these cells, maybe combined with a feed-back homeostatic mechanism that tries to keep level of hnrnph steady (Ni et al.; Genes Dev 21: ). However, when we co-transfected a plasmid containing the hnrnph cdna, we did see a slight effect on IG20/MADD splicing (Supp. Fig s5) and a clear effect on RON splicing (supp Fig s7b), which is consistent with minor changes in hnrnph abundance (not obvious by the WB analysis). What little effect we observed with hnrnph co-transfection was abrogated by the mutations in the ESSs (see lanes 4 vs. 3 in Supp Figs s5 and s7b). Together, data shown in Figures 3E and 5F (knock-down of hnrnph) and in Supp Figs s5 and s7b (co-transfection with hnrnph cdna) indicates that the hnrnph functions through the two ESSs A minor point is that the top of page 10 refers to a Figure 1F. This does not exist. Indeed, there is no splicing redirection assay for IG20. That was a mistake and we apologize. The reference was removed. Referee #2: In this interesting manuscript, the Cartegni lab describes a role for the RNA-binding protein hnrnp H in the regulation of alternative splicing of pre-mrnas associated with cancer (gliomas). There are several interesting aspects to this manuscript. First, the data clearly support a role for hnrnph in the regulation of alternative splicing leading to a pro-survival isoform of the death domain adaptor protein IG20-MADD and to the production of motility-enhancing isoform of the RON receptor tyrosine kinase. Importantly, the authors mapped the elements (ESS) responsible for the exon skipping event in both IG20 and RON. Furthermore, the authors also clearly show that hnrnp H promotes cell survival in HeLa cells and this effect is clearly mediated by inhibiting inclusion of IG20 exon 16. A particularly nice experiment is the one showing that the increased apoptosis following hnrnp H ablation can be rescued by targeted down-regulation of IG20 exon 16 (Fig. 3F). Along the same lines, LeFave and colleagues show that hnrnp H levels contribute to the invading properties in HeLa and T98G cells, and that this is at least in part due to hnrnp H- European Molecular Biology Organization 5

6 dependent modulation of RON exo n 11 splicing. Overall, my impression of this work is very positive. It nicely links a thorough characterization of cis-acting sequences and trans-acting factors required for a regulated splicing event with the development of gliomas. This work would benefit from the following revisions - In the Discussion section, the authors claim that they should test the role of hnrnp H in tumor progression in-vivo using mouse models. This is clearly beyond the scope/time-frame of this manuscript. We concur and we eliminated the speculative section from the discussion, also for the sake of length. The authors should 1- test that hnrnp H blocks cell death via MADD activity in a glioma cell line (T98G cells) as they do in HeLa (figure 3F). We agree this is an important point. We have therefore repeated the experiment in U373 glioma cells, and the results are now reported in Figure 4 (before it was in figure 3F). The results strengthen the previous data as the same close association between IG20/MADD exon 16 inclusion and cell death is maintained in the glioma cells. 2- It is shown that hnrnp H is overexpressed in gliomas as the average of several samples but the variation between samples is quite high. The authors should analyze hnrnp H levels and IG20 exon 16- lacking variants (and/or RON exon 11) levels for some individual GBM samples to demonstrate that a clear correlation exists in Glioma. We now report this analysis in Supp. Fig. 12. In the majority of the samples the two sets of data are in good agreement (20/25, including the 5 non-tumor samples), but 5/20 samples do not conform to the "high levels of hnrnph -> High level of IG20/MADD exon 16 skipping" model. One (T6) because has low level of skipping but increased hnrnph, and 4 more because on the contrary they have good exon 16 skipping but level of hnrnph comparable (or lower) then the controls. This is not surprising, especially in a tumor as heterogeneous as GBM. The observed splicing pattern could also be due to changes in expression of additional members of the hnrnph/f family that would probably result in similar effects, or in changes in other factors controlling IG20/MADD splicing (such as those recognizing the ESEs). Indeed, any one individual tumor could achieve aberrant IG20 splicing in multiple ways. However we believe our data show that -in general- hnrnph is involved in this dysregulation. Minor comments - On page 4, when referring to SF2/ASF, the author should use the new nomenclature (SRSF1). Also, it would be good to properly introduce what SR proteins are. There are several updated review articles that can be used for this. We adopted the new nomenclature throughout the paper and a short mention of the role of SR proteins and hnrnps, with two recent reviews as references, was added to the introduction. - Authors seem to be missing from the first reference "Comprehensive genomic" The reference has been corrected. Referee #3: In this work, LeFave et al. analyze the role of hnrnp H in regulating two important splicing events that occur in human gliomas and involve the IG20 and Ron genes. Overall, the work is well written and the results clear. Moreover, this work makes several points of interest regarding the multiple roles that might be played by a single splicing factor in tumor progression. European Molecular Biology Organization 6

7 1) Understandably, most of the work is focused at elucidating the role played by hnrnp H in exon 16 of IG20 and exon 11 of Ron. However, this needs to be better addressed if the authors wish to make a clearer connection with disease. For example, on page 6 the authors mention the existence of several ESE elements within IG20 exon 16. Considering the low rate of exon inclusion it is probable that they play a major role in deciding exon 16 inclusion levels. However, aside from a preliminary bioinformatics analysis that might suggest the presence of SF2, SRp40, and SRp55 binding sites (Fig.2E) no confirmation has been made at the experimental level. Is overexpression of these factors capable of substantially improving exon 11 inclusion?. Such experiments might be a worthy addition as the authors may wish to use this information to measure the amount of these proteins in their glioma samples versus controls to make sure that the drop in exon exclusion may not be also ascribed to a drop in their expression (and not just to overexpression of hnrnp H). We have now added the suggested over=expression experiments, but none of the tested SR proteins (SRSF1/SF2/ASF, SRSF5/SRp40 and SRSF6/SRp55) seem to have a significant effect on IG20/MADD exon 16 splicing (Supp Fig s2d). The factors acting through the ESEs are thus still unknown. The point that other factors might be important in disease in addition to or in alternative to hnrnph as far as these splicing events are involved is however a valid one, especially in tumors that are heterogeneous by nature. We have changed the discussion to more clearly reflect this possibility. 2) the authors were unable to detect mron expression in the mouse samples of Fig.1E. But what about the cell lines used in 1D?. If detectable, do the trend in exon 11 inclusion/skipping in Ron mimic those of exon 16 in the IG20 gene?. If so this would be an important parallelism to further support the author's hypothesis. We have now included these data. RON splicing does not varies as much as IG20/MADD in tissues, likely reflecting differences in how their splicing is regulated. However, like for IG20/MADD, also in the case of RON the skipping of exon 11 is more pronounced in cancer cell lines (consistent with previous reports). 3) In Fig.4F, the authors claim that multiple mutations in the two GGG motifs of Ron exon 11 enhance the switch towards exon inclusion. However, observing the wide variety of signals for the unspliced mrna at the top of the gel in the various lanes it is difficult to say if this is really true. Has this experiment been quantified in some way?.... Finally, what happens to exon 11 inclusion levels when hnrnp H is silenced following this minigene transfection?. The splicing of the RON minigene is inefficient probably because the very small size of the introns (87 and 80 nt) facilitate intron retention (although the endogenous gene appears to be spliced more efficiently). This makes the analysis a bit more complicated, but we believe that the effect of the mutations is very obvious (and reproducible, although with some variability in the extent of the splicing) when just the splicing product are analyzed. We have now moved the former Figure 4F to the support Information section (supp Fig s7) and substituted it with one that better represent the average result of the double mutation on RON exon 11 splicing (now Figure 5F). This panel now also shows the experiment mentioned by the reviewer in the last sentence: promotion of the minigeneís exon 11 inclusion by the concomitant knocking-down of hnrnph by sirnas (lanes 2 vs. 1). In the mutant context there is no effect, although the high basal level of inclusion of the mutant would make it difficult to appreciate. The converse experiment was also performed: as described above, co-transfection of the hnrnph cdna with the RON minigene leads to a clear induction of exon skipping in the WT but not in the double mutant, with the mentioned caveat that the levels of over-expressioní of hnrnph are not really evident by WB analysis....moreover, the authors should check again the sequences provided in the 4E inset because the Ensembl entry (ENST ) for the human Ron gene shows the presence of a final G (..TGGGCG) at the end of the sequence they provide as opposed to the one they show (..TGGGCC)... The sequence has been corrected. We thank the reviewer for catching the mistake. European Molecular Biology Organization 7

8 3) In Ghigna et al., 2005, the silencer element that they identified in Ron exon 12 corresponds to nt of this exon that also contains at least one UGGG motif and several GGG repeats. Can these sequences bind hnrnp H too?, and if so, how did the authors rule out that the effect they see in Fig.4D may not be ascribed to this silencer sequence in exon 12?. We have now looked into the contribution of exon 12 sequences, and the results are reported in Supp Fig s8. We mutated 3 GGG triplets in the exon12 silencing region and two of them do have an effect, but they mostly result in a general improvement of RON minigene splicingand a slight increase in exon 11 skipping (the opposite of what the mutations of the ESS in exon 11 do). If these are also bound by hnrnph/f (certainly a possibility), it would work by antagonizing the downstream enhancer the responds to SRSF1 (SF2.ASF). A model of how we think the regulation of RON works is reported in Supp Fig s8b. hnrnph directly inhibits exon 11 inclusions whereas the elements in exon 12 indirectly modulate this spicing events by controlling exon 12 definition. A good exon 12 definition improves the usage of the exon 12 3íss and competes out exon 11, thus resulting in exon 11 skipping, as reported. Minor points: 1) the authors suggest that hnrnp F may not play a role in regulation because its levels are unaffected by hnrnp H depletion which can by itself rescue exon 16 skipping (page 8). Nonetheless, it would be interestingto know if hnrnp F is part of the complex assembled on the TGGG elements. Have the authors tried a supershift experiment with a hnrnp F specific antibody like thay have performed for hnrnp H?. Supplementary Figure s9 shows that down-regulation of hnrnph by sirnas or by FSD-NMD has no effect on hnrnpf levels, but is associated to changes in splicing in IG20/MADD and RON. Therefore, since hnrnpf is present in those samples at normal levels (for those cells), its presence is not by itself sufficient to inhibit splicing. Naturally this does not mean that it is not or cannot be involved in regulation of these splicing event (indeed we believe it does), just that we didnít show its direct involvement (in part because of lack of specific enough reagents, such as sirnas). The entire manuscript was streamlined in order to comply to the Journal size limits notwithstanding the additional data. Therefore the specific highlight of all the changes is not practical in the main manuscript file. However, we included an additional file that contains the side-by-side comparison of the initial submission and the current revision, in case it is needed. European Molecular Biology Organization 8