Andalusian Center of Molecular Biology and Regenerative Medicine (CABIMER), Universidad de Sevilla-Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Pablo de Olavide, 41092 Seville, Spain.
Marseille Cancer Research Center (CRCM), U1068, Institut National de la Santé et de la Recherche Médicale (INSERM), UMR7258, Centre National de la Recherche Scientifique (CNRS), Aix Marseille University, Institut Paoli-Calmettes, Equipe Labellisée Ligue, 13273 Marseille, France.
Genes Dev. 2018 Jul 1;32(13-14):965-977. doi: 10.1101/gad.311274.117. Epub 2018 Jun 28.
R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA-DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.
R 环是基因组不稳定性的一个重要来源，主要是因为它们对复制进程有负面影响。Yra1/ALY 是一种从酵母到人类都大量存在的 RNA 结合因子，对于 mRNA 输出是必需的，但它的过量会导致致死和基因组不稳定。在这里，我们表明，除了 ssDNA 和 ssRNA 之外，Yra1 在体外还结合 RNA-DNA 杂交体，并且当被人为过表达时，可以以 RNA-DNA 杂交体依赖的方式被招募到染色质上，稳定 R 环并将其转化为体内的复制障碍。重要的是，Yra1 的过量会增加由转录-复制碰撞引起的 R 环介导的基因组不稳定性，无论它们是同向还是迎面碰撞。它还会导致端粒酶阴性细胞中端粒缩短并加速衰老，这与端粒复制缺陷一致。我们的结果表明，无论是否进行复制，RNA-DNA 杂交体都会在细胞中短暂形成，并且在过量的 Yra1 稳定后，会损害基因组完整性，这与 R 环介导的基因组不稳定性的两步模型一致。这项工作为理解包括肿瘤细胞在内的修复缺陷细胞中与转录相关的基因组不稳定性开辟了新的视角。
