Peer review in Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma

Decision letter
Author response

Decision letter

Chi Van Dang

Reviewing Editor; University of Pennsylvania, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma" for peer review at eLife. Your submission has been favorably evaluated by Mark McCarthy (Senior editor) and three reviewers, one of whom, Chi Dang, is a member of our Board of Reviewing Editors.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript titled, "Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma" by Grosso et al., analyzes the transcription profiles from publically available database (TCGA) of RNA-seq results from 50 ccRCC primary samples to identify improper termination events that occur in these samples. The authors identified transcriptional read-through (TRT) events in the entire genome. While this effect is noticeable it is also subtle as summarized in Figure 1C. Even though the effect occurs not exclusively in those tumors where SetD2 is mutated, grouping the tumor samples according to SetD2 mutations shows that these have an overall higher prevalence of TRT, which also correlates with higher read numbers of the adjacent gene and occurrence of chimeric transcripts. The authors conclude that mutations in the SETD2 gene, which is involved in setting H3K36 methylation along transcribed sequences, are a common feature of the ccRCC and the cause of TRT.

The same group reported previously that mutations in SETD2 are linked to reduced double-strand break repair and activation of the p53 pathway. In the current manuscript, they document a significant level of TRT in RCC, in which increased BCL2 gene expression is associated with TRT into the body of the BCL2 gene. In addition, chimeric transcripts also arise from TRT, and the fusion transcript CTSC-RAB38 is found in 20% of RCC. Using SETD2 knockout cells, the authors also documented an increase in TRT as compared with wild-type cells. Further, the authors report the formation of RNA chimeras resulting from the improper splicing of the chimeric RNAs resulting from the transcriptional read-through.

Overall, this manuscript is interesting, although their main focus, as suggested in the title, of RNA chimeras has been reported previously for ccRCC in the Nature paper published a few years ago (Nature 499,43-49 (04 July 2013) doi:10.1038/nature12222). Furthermore, a recent study in neuroblastoma revealed pervasive TRT in response to osmotic shock, which was seemingly dependent on IP3R activation (Vilborg et al. Mol Cell 2015).

Essential revisions:

While the technical aspect of this study is very well-done and the findings offer a different conceptual framework for the cancer transcriptome, they are only based on correlations and it remains unclear if they contribute to malignancy and are mechanistically linked to loss of SetD2. Without further functional evidence that the absence of SETD2 directly accounts for enhanced read-through the current work remains too preliminary. This could be achieved by knockdown of SETD2 in wild-type RCC cell lines followed by TRT measurement to determine whether loss of SETD2 function would reduce TRT. Using inducible SETD2 in SETD2 mutant RCC cell lines, one could potentially perform short term TRT measurement upon induction of SETD2 expression and/or provide colony suppression assays to document the inability to reconstitute SETD2 expression in mutant cell lines.

Minor points:

1) The authors claim that SETD2 mutations cause the improper readout using Caki-2 cell lines as a control. The Caki-2 cell line is also a ccRCC cell line, and the other lines used in this study show lower levels of Setd2 transcription. However, the authors should show that mutations in BAP, PBRM1 or VHL do not show any transcriptional read-through before arriving at that conclusion. It would be informative if the authors compared the levels of Setd2 with a cell line like HEK293, to get an idea as to how high it is expressed in Caki-2 cells relative to a 'normal' cell line.

2) The authors do not discuss or offer any explanations as to why the RNA chimera skips the last exon of the preceding gene and the first exon of the following gene. A comment on that observation would be interesting in the Discussion.

3) The authors should discuss the following points. Further work will be necessary to document whether chimeric transcripts produce functional oncoproteins and whether TRT results in transcripts that could increase the production of downstream genes, such as BCL2. The latter could be studied in future work through ribosome profiling and footprinting. Furthermore, direct evidence that a chimeric transcript (since it is unclear if these are indeed translated) has oncogenic functions will have to be studied in the future to justify the proposed model.

https://doi.org/10.7554/eLife.09214.025

Author response

Essential revisions:

Following the reviewers’ request, we now provide direct measurements of transcription read-through upon SETD2 depletion in wild-type (wt) ccRCC cells. These were achieved using two distinct approaches: (a) Analysis of RNA-seq data from SETD2 wt and SETD2-knockout ccRCC cells (Figure 3E,F) and (b) RT-qPCR analysis of transcription read-through in three different genes (MRPL23, SEL1L3 and FAM46A) following RNAi-mediated knockdown of SETD2 in a wt ccRCC cell line (Figure 3—figure supplement 3). These additional data further support our conclusion that SETD2 is necessary to prevent transcription read-through.

Using inducible SETD2 in SETD2 mutant RCC cell lines, one could potentially perform short term TRT measurement upon induction of SETD2 expression and/or provide colony suppression assays to document the inability to reconstitute SETD2 expression in mutant cell lines. Without these experiments, the manuscript would not be acceptable for publication.

To evaluate if wt SETD2 can rescue the transcription read-through phenotype, we performed single-molecule RNA FISH upon transient transfection of mutant ccRCC cell lines with wt SETD2. We used FISH probes designed against a segment of the RNA transcript encoded by the intergenic region downstream of the canonical termination site of two different genes (MRPL23 and SEL1L3, Figure 4). These new data show that reconstitution of SETD2 activity in mutant ccRCC cells rescues the transcription termination defects.

Minor points:1) The authors claim that SETD2 mutations cause the improper readout using Caki-2 cell lines as a control. The Caki-2 cell line is also a ccRCC cell line, and the other lines used in this study show lower levels of Setd2 transcription. However, the authors should show that mutations in BAP, PBRM1 or VHL do not show any transcriptional read-through before arriving at that conclusion. It would be informative if the authors compared the levels of Setd2 with a cell line like HEK293, to get an idea as to how high it is expressed in Caki-2 cells relative to a 'normal' cell line.

As we show in Figure 2C, ccRCC samples with mutations in BAP, PBRM1 or VHL do have transcription read-through. However, we show that mutations in SETD2 correlate significantly with higher levels of read-through. In the revised manuscript we now present new data showing that loss of SETD2 is sufficient to cause widespread transcription read-through in a wt ccRCC cell line. Moreover, we show that ectopic expression of SETD2 in a mutant ccRCC cell line rescues the termination defects. In addition, following the referees’ suggestion, we now include HEK293 cells in the comparative analysis shown in Figure 3A. These new data reveal that HEK293 and Caki-2 cells express SETD2 at similar levels.

We show in Figure 7B that the splicing pattern of the chimeras join the second-last exon of the upstream gene with the second exon of the downstream gene. We suggest that this pattern is in agreement with the exon definition model according to which the absence of transcription termination evicts the last 3’ss of the upstream gene favouring the use of the first 3’ss that emerges from the downstream gene. This model explains why the RNA chimeras skip the last exon of the upstream gene and the first exon of the downstream gene. We now comment this model in the Discussion.

3) The authors should discuss the following points. Further work will be necessary to document whether chimeric transcripts produce functional oncoproteins and whether TRT results in transcripts that could increase the production of downstream genes, such as BCL2. The latter could be studied in future work through ribosome profiling and footprinting. Furthermore, direct evidence that a chimeric transcript (since it is unclear if these are indeed translated) has oncogenic functions will have to be studied in the future to justify the proposed model.

These points are now discussed in the revised manuscript.

https://doi.org/10.7554/eLife.09214.026