The interplay of RNA- and Genome Surveillance in Drosophila melanogaster

• Applicant: Professor Dr. Klaus Förstemann
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 392687528

We have discovered that DNA double-strand breaks serve as initiation sites for transcription and thereby induce the formation of corresponding siRNAs in Drosophila. A genome-wide screen for the factors required to generate the response at a DNA break has yielded a large number of proteins with a role in splicing and mRNA maturation, suggesting a link between RNA and genome surveillance. We could validate this observation with cas9 induced chromosomal breaks: Upstream introns greatly stimulated siRNA production, while breaks before the first intron or in intron-less genes generated only a rather low level of siRNAs. Conflicting reports have been published on which RNA polymerase (RNA pol II or pol III) is producing the dilncRNA and our efforts help to resolve this important question. During the first funding period, we have established nascent RNA sequencing in combination with polymerase-specific immunoprecipitation and site-specific induction of DNA breaks by CRISPR/cas9. We could detect antisense-oriented and RNA polymerase II associated nascent RNAs that were more abundant upon a corresponding genomic cut. In contrast, the RNA polymerase III specific nascent RNA sequencing did not reveal any activity at the DNA break. Consistent with the much lower damage-induced siRNA yield, we could not detect any nascent RNA initiating at a break within an intron-less gene. This argues that 1) it is RNA polymerase II that transcribes the dilncRNA in Drosophila and 2) that the rate of antisense transcription, rather than downstream events such as dsRNA formation, is limiting for damage-induced siRNA generation. The outstanding questions in the field are now to refine the mechanistic link between splicing and dilncRNA transcription, substantiate mechanistic insight into how transcription / repair conflicts are resolved and whether the local chromatin structure directs and/or responds to the damage-induced transcription. We discovered a DNA-damage stimulated interaction between the Zn2+-finger protein Putzig (a confirmed hit in our screen) and the Prp19 complex of the spliceosome. Since certain other members of Putzig-containing complexes were also identified in our screen, I propose to focus the current research proposal on these factors. We will use RNAi-mediated depletion, followed by DNA cleavage and deep sequencing, to measure effects on break-derived siRNA generation directly. We will also study the DNA-damage dependent interactions with the spliceosome via co-IP and Western blotting. Chromatin-IP will be used to demonstrate recruitment of these factors, and potentially the resulting epigenetic modification, to specifically induced DNA breaks. Finally, we will employ established and newly implemented DNA repair assays to describe DNA repair phenotypes resulting from the depletion of Putzig and associated factors.

• DFG Programme: Research Grants