A minimal sequence motif drives selective tRNA dihydrouridylation by hDUS2.
A minimal sequence motif drives selective tRNA dihydrouridylation by hDUS2.
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最小序列基序驱动 hDUS2 选择性 tRNA 二氢尿苷化。
DOI：
10.1101/2023.11.04.565616
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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：
Ji,Jingwei;Yu,NathanJ;Kleiner,RalphE
通过二氢尿苷合酶（DUS）将尿苷转录后还原为二氢尿苷（D）是RNA生物学中最普遍的转化之一。在tRNA的多个位点发现了D，并且在酵母中的研究已经提出，四种真核DUS酶中的每一种修饰不同的位点，然而这种精致选择性的分子基础是未知的，并且人类DUS酶在很大程度上仍然未被表征。在此，我们研究了人二氢尿苷合酶2（hDUS 2）的底物特异性，使用基于机制的交联与5-溴尿苷（5-BrUrd）修饰的寡核苷酸探针和体外二氢尿苷化测定。我们发现，hDUS 2修改U20在D环的不同tRNA底物，并确定一个最小的GU基序内的tRNA三级折叠所需的指导其活动。此外，我们使用我们的基于机制的平台来筛选hDUS 2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的DUS修饰底物的原理，并提供了一个通用的平台，研究RNA修饰酶与序列定义的活性为基础的探针。
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 crosslinking with 5-bromouridine (5-BrUrd)-modified oligonucleotide probes andin vitrodihydrouridylation assays. We find that hDUS2 modifies U20 in the D loop of diverse tRNA substrates and identify a minimal GU motif within the tRNA tertiary fold required for directing its activity. Further, we use our mechanism-based platform to screen small molecule inhibitors of hDUS2, a potential anti-cancer target. Our work elucidates the principles of substrate modification by a conserved DUS and provides a general platform to studying RNA modifying enzymes with sequence-defined activity-based probes.