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About the Authors:
Karin Merk
Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität München, München, Germany
Marco Breinig
Affiliation: Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Im Neuenheimer Feld 580, Heidelberg, Germany
Romy Böttcher
Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität München, München, Germany
Stefan Krebs
Affiliation: Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universität München, München, Germany
Helmut Blum
Affiliation: Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universität München, München, Germany
Michael Boutros
Affiliation: Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Im Neuenheimer Feld 580, Heidelberg, Germany
Klaus Förstemann
* E-mail: [email protected]
Affiliation: Gene Center and Dept. of Biochemistry, Ludwig-Maximilians-Universität München, München, Germany
ORCID http://orcid.org/0000-0003-3641-6342Abstract
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 DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila 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 the 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 signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster....