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Interconnection between R-loops and co-transcriptional alternative splicing

Periodic Reporting for period 1 - RLOOP-AS (Interconnection between R-loops and co-transcriptional alternative splicing)

Reporting period: 2015-07-01 to 2017-06-30

Gene expression is an extremely complex mechanism involving several cellular machineries and sub-mechanisms. One of these mechanisms, called alternative splicing, is able to produce different proteins from one unique gene and understanding its regulation is of utmost importance, as mutations in its components are responsible for diseases and cancers. Notably, it has been demonstrated that UV light deregulates alternative splicing of genes important for cell survival and this might be responsible for skin cancer progression. However, the mechanistic insights remain unknown and deciphering them will help for the development of new therapeutic approaches.
It has also been shown that mutations in several splicing factors are able to stabilize R-loop formation, which happens when the nascent RNA invade the DNA duplex before the formation of the double helix. The single stranded DNA strand might then be attacked by damaging agents creating genome instability. However, if persistent R-loop formation could be a threat for the genome integrity, the controlled formation of R-loop was recently shown to regulate gene expression. The aim of this project was to study the possible connection between R-loop formation and alternative splicing regulation, especially after DNA damage induction, and to understand the underlying mechanisms of regulation. We focused on RNA polymerase II kinetics and epigenetic modifications, as previous studies linked these two mechanisms with alternative splicing regulation.
We have shown that after DNA damage, notably induced by UV light, R-loops were quickly formed, before alternative splicing was affected and that removal of R-loops could prevent alternative splicing alterations, suggesting a regulatory role of R-loops after DNA damage.
We have accumulated a number of evidences that suggest a connection between R-loop formation and alternative splicing regulation, which was not expected before starting this project. With the use of cutting edge techniques, such as RNA sequencing of nuclear RNA, we have identified a repertoire of deregulated alternative splicing events after DNA damage induction, which characteristics helped us to understand the underlying mechanisms. In fact, computational analysis, using a package dedicated to alternative splicing developed in the lab, indicated that the deregulated events were enriched in GC-rich regions, the main condition of R-loop formation.
Then, we have validated that R-loops were stabilized after DNA damage using super resolution microscopy. For the first time, we showed that R-loops formed in the nucleus, when other publications using confocal microscopy were unable to show it. Moreover, we showed that R-loops are quickly formed after DNA damage, after 5 min, when alternative splicing, on chromatin RNA, is affected after around 45 min – 1 hour, depending on transcription rate, suggesting a possible role for R-loop formation in alternative splicing regulation.
Using minigene based experiments, we showed that R-loop were stabilized on a DNA template which mRNA suffered alternative splicing deregulation. Moreover, the removal of R-loop by an enzyme that specifically cleaves this structure could reverse this effect, indicating a role of R-loops in alternative splicing regulation after DNA damage. Next, we precisely mapped R-loop formation genome wide by developing a new technique, based on the immunoprecipitation of R-loops using a specific antibody and sequencing the RNA caught in the R-loop. Their localization suggested a possible role in alternative splicing regulation genome wide, as R-loops were enriched close to many alternative splicing events. We are currently investigating by which mechanism R-loops regulate alternative splicing.
These results were presented as an oral presentation at the 2017 RNA meeting in Prague and at the EMBO 3’ end processing in eukaryotic genomes meeting in Oxford
At the beginning of the project, no evidence of regulation of alternative splicing by R-loops was demonstrated, and few groups were working on R-loops. Nowadays, this field has increased significantly, showing the importance of this RNA/DNA hybrid structure on gene expression regulation and cancer development. This project allowed us to make some pioneering and innovative discoveries in the regulation of alternative splicing after DNA damage. For example, we are the first ones using super resolution microscopy to detect R-loop formation. While many studies showed confocal images where it is hard to see a nuclear stabilization of R-loops, the use of this new technology allowed us to demonstrate a clear R-loop formation in the nucleoplasm after DNA damage. Moreover, we develop a new technique to precisely map R-loop genome wide with a better resolution than the existing method, by sequencing the RNA caught in the R-loops. This technique allows us to also detect the strand specificity, whereas this was impossible before. Taken together, these techniques allowed us to demonstrate a clear role of R-loops in alternative splicing regulation after DNA damage, bringing a new – and early - player at the table. We hope to fully decipher the mechanistic insights of this process, which will allow the development of new therapeutic strategies or early detection of skin cancers, for example.
We are now focusing on how R-loops can alter alternative splicing regulation. We focused on two hypotheses: RNA polymerase II kinetics and epigenetic modifications. As some DNA damaging agents are known to induce RNA polymerase II hyperphosphorylation, we decided to perform some mNET-seq experiments to understand how RNA polymerase II phosphorylation pattern could affect R-loops or vice versa. For the second hypothesis, R-loops were shown to be able to induce chromatin structure modifications. We are developing the nChIP-seq technique to map precisely epigenetic marks and we hope to find local chromatin structure modification where R-loops are formed and alternative splicing affected.