Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome
Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome
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化学下拉揭示了哺乳动物转录组的动态假尿苷化
DOI：
10.1038/nchembio.1836
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发表时间：
2015-08-01
影响因子：
14.8
通讯作者：
Yi, Chengqi
中科院分区：
文献类型：
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作者：
Li, Xiaoyu;Zhu, Ping;Yi, Chengqi
假尿苷（psi）是最常见的RNA转录后修饰，但其在mRNA中的发生率、作用机制和功能尚不清楚。在这里，我们进行了定量MS分析，并表明psi是更普遍的（psi/U比类似于0.2-0.6%）在哺乳动物的mRNA比以前认为的。我们开发了N-3-CMC富集的假尿苷测序（CeU-Seq），这是一种选择性化学标记和下拉方法，用于识别1，929个人类转录本中的2，084 psi位点，其中4个（在核糖体RNA和EEF 1A 1 mRNA中）经过生物化学验证。我们发现，hPUS 1，一个已知的psi合酶，作用于人的mRNA;在压力下，CeU-Seq表现出诱导型和压力特异性mRNA假尿苷酸化。将CeU-Seq应用于小鼠转录组揭示了保守的和组织特异性的假尿苷酸化。总的来说，我们的方法可以全面分析转录组范围内的假尿苷酸化，并提供工具的PSI介导的表观遗传调控的功能研究。
Pseudouridine (psi) is the most abundant post-transcriptional RNA modification, yet little is known about its prevalence, mechanism and function in mRNA. Here, we performed quantitative MS analysis and show that psi is much more prevalent (psi/U ratio similar to 0.2-0.6%) in mammalian mRNA than previously believed. We developed N-3-CMC-enriched pseudouridine sequencing (CeU-Seq), a selective chemical labeling and pulldown method, to identify 2,084 psi sites within 1,929 human transcripts, of which four (in ribosomal RNA and EEF1A1 mRNA) are biochemically verified. We show that hPUS1, a known psi synthase, acts on human mRNA; under stress, CeU-Seq demonstrates inducible and stress-specific mRNA pseudouridylation. Applying CeU-Seq to the mouse transcriptome revealed conserved and tissue-specific pseudouridylation. Collectively, our approaches allow comprehensive analysis of transcriptome-wide pseudouridylation and provide tools for functional studies of psi-mediated epigenetic regulation.