DNA:RNA hybrids or R-loops are formed by pairing of transcripts with a complementary DNA strand. R-loops form at transcriptional pause sites, termination regions of genes and at repetitive regions. Aberrant accumulation of R-loops can lead to DNA damage and genome instability, promoting carcinogenesis. Our interest in R-loops was prompted by our studies on the TGS1 hypermethylase, which controls the biogenesis of small nuclear RNAs (snRNAs), the fidelity of splicing and transcription termination in human cells. We also found that TGS1 and Topoisomerase 2 (Top2) physically interact in Drosophila and that their loss causes R-loop accumulation and cell death in the eye imaginal discs. TGS1 and Top2 are overexpressed in tumors with poor prognosis and may therefore protect cancer cells against excessive R-loops that would cause crisis.
We plan to explore the role of TGS1 in R-loop formation and genome stability, by determining the effects of TGS1 depletion on genome-wide transcription patterns in both HeLa cells and Drosophila using both Illumina and Nanopore sequencing. We will also map R-loops in both the human and fly genome and screen for modifiers of the Tgs1-dependent phenotype in Drosophila. Finally, we plan to characterize additional Drosophila genes involved in transcription and whose deficiency in embryos results in DNA damage to explore whether these lesions correlate with R-loop accumulation.
Our experiments should elucidate whether the genes that prevent co-transcriptional stress also prevent R-loop formation and DNA damage, and provide insight into the cascade of events that lead to aberrant accumulation of R-loops.
Most studies on the speed and efficiency of splicing and how splicing perturbation affects the accumulation of R-loops, were performed in cells depleted of the splicing factor SF3B1. Our proposal exploits the TGS1 mutant background, which is particularly convenient, as TGS1 mutant cells have a strong enrichment of different classes of aberrant transcripts and thus are particularly suited to identify even very rare molecules. Our high-resolution characterization of the aberrant transcripts generated upon TGS1 depletion, combined with the fine mapping of R-loops accumulations in the genome, will give us a unique opportunity to explore at high depth the consequences of defective splicing on genome stability. The joint use of Illumina and ONT sequencing is a recent approach, which will help us ascertaining whether aberrant transcripts, which contain both splicing defects and extended 3¿UTRs, are generated in TGS1-depleted cells. These aberrant molecules will allow a deeper understanding of the interplay between splicing and termination and provide valuable information on how these processes are coupled.
By exploiting the power of Drosophila genetics and the use of live microscopy studies of early embryo development, we will have the opportunity to characterize new functions, which regulate transcriptional regulation in a context that is particularly vulnerable to transcriptional stress and R-loops accumulation.
In vivo analyses on embryos depleted of factors involved in different aspects of RNA metabolism will unravel the dynamics of R-loops formation and behavior during the early nuclear divisions and monitor the recruitment of processing factors on chromosomes that accumulate R-loops. Importantly, analyses on embryos that exhibit chromosome bridges will help us deciphering the different mechanisms underlying bridge formation and will also allow us to establish a correlation between R-loops and chromosome damage (bridges and breaks). The fact that chromosome bridges and breaks are observed in mutants defective in transcription does not necessarily imply that these aberrations are the consequence of abnormal transcription. Our experiments will ascertain which type of lesions are generated by R-loops accumulation and whether these lesions are dependent on defective transcription.