CisTransRloop

Several threats endanger the tight equilibrium between genome maintenance and expression that guarantees nuclear homeostasis. Among them, the formation of R-loops through hybridization of nascent mRNAs onto their DNA templates has emerged as a major cause of transcription-associated genetic instability. However, the molecular mechanisms involved in R-loop sensing and processing remain largely undiscovered. In particular, the cis- and trans-acting factors that influence the fate of R-loops in space and time remain to be deciphered. Based on unique preliminary results and approaches gathered by our consortium, this project will explore the impact of R-loops on genome dynamics, using mainly budding yeast as a model system. 1. We will investigate how R-loops affect the spatial organization of the genome. We have found that R-loop forming loci are repositioned in the nucleus in a R-loop-dependent manner. We will identify the molecular mechanisms at play and investigate their impact on R-loop genotoxicity. For this purpose, we will perform genome-wide analyses of gene localization and R-loop levels. We will further interfere with R-loop accumulation and analyze the consequences for gene localization, using microscopy and biochemical approaches. Finally, we will artificially modulate the localization of R-loop-forming loci and evaluate the impact on R-loop-associated genetic instability. 2. We will decipher how R-loops contribute to genome mutability and molecular evolution. To understand why only certain R-loops are genotoxic, we will profile the mutations they trigger through genome-wide analyses in R-loop-accumulating mutants. The genomic features that determine the genotoxic potential of R-loops will be further identified through correlation analyses and validated using specific R-loop-forming reporters. 3. We will identify which trans-acting factors modulate these processes and impinge on R-loop-dependent genetic instability. We will use innovative genetic screens in an attempt to systematically identify the cellular factors that contribute to R-loop-dependent processes, and characterize their mode of action using dedicated molecular and biochemical assays. Their conservation will be analyzed in the frame of a collaboration. To achieve these different aims, this consortium combines unique and complementary skills in molecular biology, genomics and microscopy. The realization of this project is thereby expected to shed light on this emerging field of research, and to provide clues for the understanding of the multiple pathological situations to which R-loops have been associated.