Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae.
Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae.
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DOI：
10.1371/journal.pone.0110799
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Brown PO
中科院分区：
文献类型：
--
作者：
Lovejoy AF;Riordan DP;Brown PO
我们开发了一种新的技术，称为假尿苷位点识别测序（PSI-seq），用于基于CMCT处理后特异性在假尿苷处诱导的逆转录终止的细胞RNA的单碱基分辨率的假尿苷化位点的转录组范围的映射。来自S.酵母18 S和25 S核糖体RNA中的43个已知假尿苷都能被酿酒酵母特异性地检出。此外，PSI-seq在酵母转录组中的应用揭示了数十种mRNA中存在位点特异性假尿苷化，包括RPL 11 a，TEF 1和其他与翻译有关的基因。为了确定mRNA假尿苷化的机制，我们从基因上删除了候选的假尿苷合酶（Pus）酶，并在体外重建了它们的活性。这些实验表明，Pus 1酶是RPL 11 a mRNA假尿苷化所必需和充分的，而Pus 4修饰TEF 1 mRNA，Pus 6假尿苷化KAR 2 mRNA。最后，我们确定在来自相关酵母S. TEF 1 mRNA中的E11 - 239在S. mikitae和S. pombe，表明这些pseudouridylation是古老的，进化上保守的RNA修饰。这项工作确立了真核生物mRNA的位点特异性假尿苷化是一种遗传程序化的RNA修饰，其通过不同的机制天然地发生在多种酵母转录物中，这表明mRNA假尿苷化可能提供重要的新型调节功能。我们在这里报告的方法和策略应该普遍适用于发现假尿苷化，或其他RNA修饰，在不同的生物学背景。
We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq), for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus) enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.
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RNA 假尿苷化：对旧修饰的新见解。
影响因子：
13.8
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