Cell type-specific translational regulation by human DUS enzymes.
Cell type-specific translational regulation by human DUS enzymes.
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人类 DUS 酶的细胞类型特异性翻译调节。
DOI：
10.1101/2023.11.03.565399
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Kleiner,RalphE
中科院分区：
文献类型：
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作者：
Yu,NathanJ;Dai,Wei;Li,Ang;He,Muhan;Kleiner,RalphE
二氢尿苷是tRNA上存在的丰富且进化上保守的修饰核苷，但缺乏哺乳动物中个体修饰位点和相关DUS书写酶的表征和功能研究。在这里，我们使用的化学探测策略，RNABPP-PS，以确定5-氯尿苷作为一个通用的活动为基础的探针人类DUS酶。我们使用基于机制的RNA-蛋白质交联并通过化学转化和突变分析来绘制D修饰图，以揭示人类tRNA上D修饰位点的景观。此外，我们敲除两个模型人类细胞系中的单个DUS基因，以研究tRNA表达水平和密码子特异性翻译延伸的调节。我们表明，虽然大多数tRNA物种中都存在D修饰，但D的缺失仅以细胞类型特异性的方式扰乱了一部分tRNA的翻译功能。我们的工作为研究不同生物系统中的D和DUS酶提供了强有力的化学策略，并深入了解了普遍存在的tRNA修饰在翻译调控中的作用。
Dihydrouridine is an abundant and evolutionary conserved modified nucleoside present on tRNA, but characterization and functional studies of individual modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as a general activity-based probe for human DUS enzymes. We map D modifications using mechanism-based RNA-protein crosslinking and through chemical transformation and mutational profiling to reveal the landscape of D modification sites on human tRNAs. Further, we knock out individual DUS genes in two model human cell lines to investigate regulation of tRNA expression levels and codon-specific translation elongation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell type- specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.