rRNA transcription is integral to phase separation and maintenance of nucleolar structure.
rRNA transcription is integral to phase separation and maintenance of nucleolar structure.
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DOI：
10.1371/journal.pgen.1010854
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发表时间：
2023-08
期刊：
影响因子：
4.5
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中科院分区：
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核糖体 RNA 聚合酶 (Pol) I 在核仁中转录核糖体 RNA (rRNA) 对于核糖体生物发生是必需的，而核糖体生物发生与细胞生长和增殖密切相关。核糖体生物发生的扰动导致与核仁结构改变相关的称为核糖体病的组织特异性疾病。然而，rRNA 转录和核糖体生物发生如何在正常发育和疾病发病机制中调节核仁结构仍知之甚少。在这里，我们发现 rRNA 转录和核糖体生物合成所需的 Pol I 亚基中的纯合无效突变会导致植入前致死。此外，我们发现 Polr1a-/-、Polr1b-/-、Polr1c-/- 和 Polr1d-/- 突变体的核仁结构存在缺陷，核仁前体数量减少和核仁体积随之增加，从而导致单个浓缩核仁。植入前和妊娠中期胚胎以及 hiPSC 中 Pol I 的药理学抑制同样会导致单个浓缩核仁或碎片核仁。我们发现，当 Pol I 功能和 rRNA 转录受到抑制时，核仁颗粒区室的粘度增加，这破坏了其相分离特性，导致单个浓缩核仁。然而，如果细胞进行有丝分裂，rRNA 转录的缺失会阻止核仁的重新组装，并表现为核仁碎片。综上所述，我们的数据表明，Pol I 功能和 rRNA 转录是发育和疾病发病机制中维持核仁结构和完整性所必需的。核糖体 RNA (rRNA) 是核糖体的催化成分，在每个细胞中将信使 RNA 翻译成蛋白质。 rRNA 的转录过程发生在无膜核间细胞器（核仁）中，当核仁受到破坏时，会导致组织特异性缺陷，即人类核糖体病。 rRNA 转录的破坏如何影响核仁结构尚不清楚。我们生成了四种不同的小鼠 RNA 聚合酶 (Pol) I 复合物亚基突变体，该复合物转录 rRNA。破坏 rRNA 转录会导致植入前胚胎死亡。在致死之前，突变体胚胎表现出 rRNA 减少和一个大的浓缩核仁，而对照胚胎中只有 2-5 个核仁。在带有不同核仁区室标记的人诱导多能干细胞 (hiPSC) 中，通过药理学抑制 rRNA 转录，会产生类似于 Pol I 亚基小鼠突变体的浓缩核仁或片段化核仁。细胞核仁的命运取决于其在 Pol I 抑制之前所处的细胞周期阶段。总而言之，我们的数据表明，Pol I 介导的 rRNA 转录对于发育过程中维持核仁结构和完整性至关重要，但会因疾病而受到破坏。
Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease. Ribosomal RNA (rRNA) is the catalytic component of a ribosome, which translates messenger RNA into protein in every cell. The process of transcribing rRNA takes place in a membraneless internuclear organelle, the nucleolus, which when disrupted leads to tissue-specific defects known as ribosomopathies in humans. How disruption of rRNA transcription affects nucleolar structure is poorly understood. We generated four different mouse mutants of subunits of the RNA Polymerase (Pol) I complex, which transcribes rRNA. Disrupting rRNA transcription results in pre-implantation embryo lethality. Prior to lethality, the mutant embryos exhibit reduced rRNA and one large, condensed nucleolus, compared to 2–5 nucleoli in control embryos. Pharmacologically inhibiting rRNA transcription in human induced pluripotent stem cells (hiPSCs) tagged with labels for different compartments of the nucleolus results in either a condensed nucleolus similar to the Pol I subunit mouse mutants or fragmented nucleoli. The fate of the nucleolus in a cell depends on the phase of cell cycle it is in prior to Pol I inhibition. Altogether, our data suggests that Pol I mediated rRNA transcription is vital for maintaining nucleolar structure and integrity during development and is disrupted in association with disease.