Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [ChIP-seq]

Post-transcriptional modifications of mRNA have emerged as novel regulators of gene expression. Pseudouridylation is the most abundant and widespread type of RNA modification in living organisms; however, the biological role of pseudouridine in mRNAs remains poorly understood. Here, we show that the pseudouridine synthase dyskerin associates with RNA polymerase II and is enriched at RNA polymerase II-transcribed genes genome-wide. As part of the H/ACA complex, dyskerin binds to thousands of mRNAs and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a fully complementary guide RNA. In cells lacking dyskerin, pseudouridylation of mRNAs is reduced, while at the same time, de novo protein production is enhanced, indicating that pseudouridylation of mRNAs by dyskerin interferes with translation. Accordingly, pseudouridylation of an mRNA by dyskerin in vitro results in reduced translation of that mRNA. Moreover, in patients with dyskeratosis congenita caused by inherited mutations in the DKC1 gene, binding between mutated dyskerin and mRNAs is altered and pseudouridylation of mRNAs severely reduced. Our findings reveal a new critical function of the H/ACA complex in directing translation control with important implications for development and disease. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RNAPII and dyskerin in U2OS and HCT116 cells.

信使RNA（mRNA）的转录后修饰已被证实为基因表达的新型调控因子。假尿苷酸化是生物体内最丰富、分布最广泛的RNA修饰类型，但目前学界对mRNA中假尿苷的生物学功能仍知之甚少。本研究发现，假尿苷合酶dyskerin可与RNA聚合酶II（RNA polymerase II）结合，并在全基因组范围内的RNA聚合酶II转录基因区域富集。作为H/ACA复合物的组成部分，dyskerin能够结合数千条mRNA并负责其假尿苷酸化，该修饰过程发生在染色质中，且似乎无需完全互补的向导RNA。在缺失dyskerin的细胞中，mRNA的假尿苷酸化水平显著降低，与此同时新生蛋白质的合成效率却有所提升，这表明dyskerin介导的mRNA假尿苷酸化会干扰翻译过程。相应地，体外实验中由dyskerin介导的mRNA假尿苷酸化会导致该mRNA的翻译效率下降。此外，在因DKC1基因遗传性突变引发先天性角化不良症的患者体内，突变型dyskerin与mRNA的结合发生异常，且mRNA的假尿苷酸化水平大幅降低。本研究揭示了H/ACA复合物在调控翻译过程中的全新关键功能，这一发现对发育与疾病研究具有重要意义。
实验设计：在U2OS与HCT116细胞系中针对RNA聚合酶II（RNAPII）及dyskerin开展染色质免疫共沉淀测序（ChIP-seq）。