Splicing stimulates siRNA formation at < i > Drosophila < /i > DNA double-strand breaks

by Karin Merk, Marco Breinig, Romy B öttcher, Stefan Krebs, Helmut Blum, Michael Boutros, Klaus Förstemann DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DN A ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in culturedDrosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene ’s first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at t he targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a sign aling hub. We conclude that convergent transcription...
Source: PLoS Genetics - Category: Genetics & Stem Cells Authors: Source Type: research