GTOXOME · The impact of genotoxic stress on the mRNA-interactome and RNA-regulons

The impact of genotoxic stress on the mRNA-interactome and RNA-regulons

RNA-binding proteins (RBP) play critical roles in stress responses like DNA damage through interactions with elements within functionally related RNAs, called regulons. One of the most cytotoxic forms of DNA damage are DNA double strand breaks (DSBs) that can lead to mutagenic events and, when improperly repaired, to cell death. This projects aims to globally identify RNA-binding proteins, which are critical in the cellular DNA damage response. Furthermore, selected candidates are functionally characterized for their implication in DNA damage repair and /or cell death mechanisms. By combining RNA-interactome capture with comparative quantitative mass spectrometry, we uncovered over 40 RBPs that respond to genotoxic stress (no stress, low DNA damage, and severe DNA damage) by increasing or decreasing their RNA-binding activities. From these proteins half are already known RBPs. Among regulated previously unknown RBPs we identified the selective autophagy receptor p62, also known as the multifunctional protein Sqstm1. We could not only validate p62 as a novel RBP, but also demonstrate its increased RNA-binding activity upon DNA damage and other forms of stress. To assess the functionality of p62 as a genotoxic stress related RBP, we made use of the CRISPR/Cas9 genomic engineering technique to generate p62 knockout (ko) NIH-3T3 (murine fibroblast) cells. This system, in combination with an inducible p62 re-introduction, was established as a rescue system to access the physiological significance of p62 as a RBP in the DNA damage response. This cellular tool-set allowed a comprehensive analysis in phenotypical assays. In comparison to control cells, the p62 ko cells demonstrate a reduced proliferation rate under non-stressed conditions, whereby an accompanied increase in cell death could not be verified. Cell cycle analysis of synchronized cells via FACS revealed a lowered cell cycle rate of the p62ko cells paired with an increase in the overall G2 phase population, suggesting an inaccuracy in G2-checkpoint resumption. This phenotype could be rescued by reintroducing p62 in the ko cells, emphasizing p62´s role in G2-checkpoint resumption. Furthermore, we investigated the effect of p62 ko on cells responses to DNA-damage induction. After y-irradiation p62 ko cells show no change in fast response (1h post DSB induction) measured by DSB amount and 53bp1 foci as indicators for early DNA damage repair. Interestingly, 24h post DSB induction, when DNA damage repair is mainly successfully completed in control cells, the p62 ko cells display an increased amount of micronuclei and 53bp1 bodies. Both are known genomic instability phenotypes, indicating a checkpoint defect. Inducible p62 rescue experiments in the ko cells, again reduce the number of micronuclei and 53bp1 bodies after y-irradiation, indicating a role of p62 in genomic instability upon DSB induction. Taken together, this project identified over 40 RBPs involved in the DNA damage response. Among these proteins p62 was validated for its previously uncharacterized RNA-binding function and being involved in the genotoxic stress response.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз