The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated “promoter and pre-rRNA antisense” RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

中文翻译：

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复制蛋白 A 与核仁 R 环结合并调节 rRNA 转录和核仁形态

核仁是一个重要的细胞区室，其中核糖体 RNA (rRNA) 被转录，并且某些对细胞生长至关重要的应激通路在其中被协调。在这里，我们报告了 DNA 复制和修复因子复制蛋白 A (RPA) 在控制核仁稳态中的新功能。我们表明 DNA:RNA 解旋酶 senataxin (SETX) 的丢失促进了 RPA 核仁定位，并且这种重新定位取决于 R 环的存在。值得注意的是，这种核仁 RPA 表型也在喜树碱 (CPT) 诱导的基因毒性应激以及 SETX 缺陷型 AOA2 患者成纤维细胞中观察到。扩展这些结果，我们发现 RPA 在 CPT 处理后被招募到 rDNA，其中 RPA 可防止 R 环诱导的 DNA 双链断裂。此外，我们发现 RPA 的缺失显着降低了 47S pre-rRNA 水平，这伴随着 RNAP II 介导的“启动子和前 rRNA 反义”RNA 以及 RNAP I 转录的基因内间隔 RNA 的表达增加。最后，很可能反映了上述情况，我们发现 RPA 的缺失促进了核仁结构的解体，其特征是出现尺寸减小的核仁。我们的研究结果既表明了 RPA 通过 pre-rRNA 转录控制在核仁中发挥的新作用，也强调了 RPA 在核仁稳态中的功能与生理和病理条件下的 R-loop 分辨率有关。并且可能反映了上述情况，我们发现 RPA 的缺失促进了核仁结构的解体，其特征是出现尺寸减小的核仁。我们的研究结果既表明了 RPA 通过 pre-rRNA 转录控制在核仁中发挥的新作用，也强调了 RPA 在核仁稳态中的功能与生理和病理条件下的 R-loop 分辨率有关。并且可能反映了上述情况，我们发现 RPA 的缺失促进了核仁结构的解体，其特征是出现尺寸减小的核仁。我们的研究结果既表明了 RPA 通过 pre-rRNA 转录控制在核仁中发挥的新作用，也强调了 RPA 在核仁稳态中的功能与生理和病理条件下的 R-loop 分辨率有关。