Role of small non-coding RNAs in DSB repair in Arabidopsis thaliana

Abstract

DNA double-strand breaks (DSB) are highly toxic lesions that must be repaired to avoid major losses of genetic materials or genome rearrangements. A number of highly conserved molecular pathways are involved in DSB repair, each resulting in different repair outcomes for a given DSB. Over the past decade, accumulating evidence has revealed the emergence of an unexpected new actor in DSB repair: RNA. Notably, small RNAs have been observed at DSB-flanking sequences and hypothesized to be involved in their repair. However, these DSB-induced small RNAs (diRNAs) were so far observed in studies using reporter systems, and their natural occurrence as well as their role in DNA repair remains debated. To address this question, we investigated diRNA biogenesis in Arabidopsis thaliana using CRISPR/Cas12a to induce targeted DSBs at three actively transcribed loci and one untranscribed locus. We found that 21-nt diRNAs are generated at transcribed loci, in a Dicer-like 4 (DCL4) dependent manner, but not at the untranscribed locus, indicating that active transcription is required for diRNA production. Remarkably, long-read DNA sequencing analysis shows that these diRNAs do not detectably influence DSB repair via either end-joining or microhomology-mediated end-joining. Furthermore, endogenous diRNAs have a modest effect on homologous recombination-associated repair, as impairment of the diRNA pathway in ago2 mutants resulted in only a slight reduction in RAD51 focus formation following Cas12a-induced DSBs. Together, these findings indicate that diRNAs are not essential for DSB repair itself but may function in the post-transcriptional silencing of damaged genes, likely to prevent aberrant transcription until proper repair is completed.