Biology subjects

Verbeke, J.

Publications and source records attributed to Verbeke, J..

3 recordsLinked to original sources

bioRxiv2026

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

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.

genetics↗

bioRxiv2024

Context effects on repair of 5'-overhang DSB induced by Cas12a in Arabidopsis.

Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much-studied Cas9 nuclease cleaves DNA to generate blunt-ended, double-strand breaks (DSB), but relatively less is known about the repair of other types of breaks, such as those with 5-overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by End-Joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSB generate deletions at one or both cut-sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single-cut and double-cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of an alternative class of repair events, corresponding to highly efficient repair by Single-strand Annealing recombination.

genetics↗

bioRxiv2022

BNIP3L-mediated mitophagy triggered by Brucella in host cells is required for bacterial egress

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers a strong mitochondrial network fragmentation accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles in the late steps of cellular infection. The expression of the mitophagy receptor BNIP3L induced by B. abortus is essential for these events and relies on the iron-dependent stabilization of the hypoxia-inducible factor 1 alpha. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit of the host cell as BNIP3L depletion drastically reduced the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

microbiology↗