Sequence- and Structure-Specific tRNA Dihydrouridylation by hDUS2.
Sequence- and Structure-Specific tRNA Dihydrouridylation by hDUS2.
复制标题
hDUS2 进行序列和结构特异性 tRNA 二氢尿苷化。
DOI：
10.1021/acscentsci.3c01382
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发表时间：
2024
影响因子：
18.2
通讯作者：
Kleiner,RalphE
中科院分区：
文献类型：
--
作者：
Ji,Jingwei;Yu,NathanJ;Kleiner,RalphE
二氢吡啶合成酶（DUS）将尿嘧啶转录后还原为二氢吡啶(D)是RNA生物学中最普遍的转化之一。D存在于trna的多个位点，在酵母中的研究表明，四种真核生物DUS酶中的每一种都修饰了不同的位点；然而，这种精细选择性的分子基础是未知的，人类DUS酶在很大程度上仍未被表征。本研究利用5-溴吡啶修饰的寡核苷酸探针和体外二氢尿嘧啶化实验，研究了人二氢尿嘧啶合成酶2 （hDUS2）的底物特异性。我们发现hDUS2在不同的tRNA底物上特异性地修饰U20，并在tRNA D环中鉴定出一个最小的GU序列，该序列是选择性底物修饰的基础。此外，我们利用基于机制的平台筛选hDUS2的小分子抑制剂，这是一种潜在的抗癌靶点。我们的工作阐明了保守DUS修饰底物的原理，并为使用基于序列定义的活性探针研究RNA修饰酶提供了一个通用平台。
The post-transcriptional reduction of uridine to dihydrouridine (D) by dihydrouridine synthase (DUS) enzymes is among the most ubiquitous transformations in RNA biology. D is found at multiple sites in tRNAs, and studies in yeast have proposed that each of the four eukaryotic DUS enzymes modifies a different site; however, the molecular basis for this exquisite selectivity is unknown, and human DUS enzymes have remained largely uncharacterized. Here we investigate the substrate specificity of human dihydrouridine synthase 2 (hDUS2) using mechanism-based cross-linking with 5-bromouridine (5-BrUrd)-modified oligonucleotide probes andin vitrodihydrouridylation assays. We find that hDUS2 exclusively modifies U20 across diverse tRNA substrates and identify a minimal GU sequence within the tRNA D loop that underlies selective substrate modification. Further, we use our mechanism-based platform to screen small molecule inhibitors of hDUS2, a potential anticancer target. Our work elucidates the principles of substrate modification by a conserved DUS and provides a general platform for studying RNA modifying enzymes with sequence-defined activity-based probes.