Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks

Abstract

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.

Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference

“…Our genome editing and deep sequencing experiments in this manuscript clearly demonstrate that a redundant RLC activity linked to Loqs exists in S2-cells. This is consistent with data from a genome-wide screen of DNA-damage induced siRNA generation where only loqs , but not r2d2 , was recovered as a candidate ( 66 ). Is the alternative RLC that we describe in S2-cells a particular idiosyncrasy of this cell line?…”

Next-generation sequencing reveals two populations of damage-induced small RNAs at endogenous DNA double-strand breaks

“…(16) would result in the generation of diRNAs mapping both downstream and upstream of the DSB. However, the diRNAs in the 28S locus show a very distinct distribution and map exclusively upstream of the I-PpoI cleavage site, which supports the idea that diRNA biogenesis relies on pre-existing sense transcripts, as previously proposed by others (13,23).…”

“…Furthermore, next-generation sequencing confirmed the production of diRNAs in plants at a transgenic reporter locus harbouring a cleavage site for an endonuclease (17). DiRNAs were also detected after Cas9-mediated cleavage in unique genomic locations in the Drosophila genome (23) and at DSBs within telomeres of mammalian cells (24). However, attempts to sequence diRNAs in mammalian cells at unique genomic loci in a natural chromatin context have been unsuccessful and their existence in genetically unperturbed cells has been questioned (14).…”

“…If this is the case, difficulties in revealing diRNAs at unique DSBs in mammalian cells could be due to their low abundance or fast turnover. Two, the extent of diRNA formation depends on local transcription and therefore only genic regions will give rise to diRNAs (13,23,26). In this scenario, diRNA production could contribute to the particular DNA repair features of transcribed genes (27) or to the degradation of aberrant mRNAs produced at the damaged gene locus, as shown in Drosophila melanogaster (23).…”

The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements

“…53BP1 is also associated with DDRNAs and dilncRNAs engaged in the mechanisms of 53BP1 focus formation [102]. Another function of DDRNAs is that they can target potentially damaged mRNA in the proximity of DSB [108]. Thus, damage response RNAs may play various functional roles in the DNA damage response.…”