{"title":"Sequence- and Structure-Specific tRNA Dihydrouridylation by hDUS2","authors":"Jingwei Ji, Nathan J. Yu and Ralph E. Kleiner*, ","doi":"10.1021/acscentsci.3c01382","DOIUrl":null,"url":null,"abstract":"<p >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 and <i>in vitro</i> dihydrouridylation 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.</p><p >We report a systematic investigation of the sequence and structural requirements for human dihydrouridine synthase 2 (hDUS2)-mediated dihydrouridylation using 5-bromouridine-modified tRNA activity probes and oligonucleotide LC-MS/MS-based analysis.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7000,"publicationDate":"2024-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.3c01382","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Central Science","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acscentsci.3c01382","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
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 and in vitro dihydrouridylation 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.
We report a systematic investigation of the sequence and structural requirements for human dihydrouridine synthase 2 (hDUS2)-mediated dihydrouridylation using 5-bromouridine-modified tRNA activity probes and oligonucleotide LC-MS/MS-based analysis.
期刊介绍:
ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.