Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks
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...
Saved in:
Published in | PLoS genetics Vol. 13; no. 6; p. e1006861 |
---|---|
Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
19.06.2017
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | 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. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks. |
---|---|
Bibliography: | new_version ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Conceptualization: KF MBo KM.Data curation: MBr SK HB KF.Formal analysis: KM KF MBr SK.Funding acquisition: HB MBo KF.Investigation: KM RB.Methodology: MBr KM HB SK KF MBo.Project administration: MBo KF.Resources: MBo SK HB.Software: KF KM.Supervision: MBr KF.Validation: KM KF.Visualization: KM KF.Writing – original draft: KM KF.Writing – review & editing: KM SK RB MBr MBo SK HB. The authors have declared that no competing interests exist. |
ISSN: | 1553-7404 1553-7390 1553-7404 |
DOI: | 10.1371/journal.pgen.1006861 |