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ABSTRACT Dihydrouridine is an abundant and conserved modified nucleoside present on tRNA, but characterization and functional studies of 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 an activity-based probe for human DUS enzymes. We map D modifications using RNA-protein crosslinking and chemical transformation and mutational profiling to reveal D modification sites on human tRNAs. Further, we knock out individual DUS genes in two human cell lines to investigate regulation of tRNA expression levels and codon-specific translation. 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. 

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 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. 

ABSTRACT Reactive small-molecule probes are widely used for RNA structure probing, however current approaches largely measure average RNA transcript dynamics and do not resolve structural differences that occur during folding or transcript maturation. Here, we present SNIPER-seq, an RNA structure probing method relying upon metabolic labeling with 2’-aminodeoxycytidine, structure-dependent 2’-amino reaction with an aromatic isothiocyanate, and high-throughput RNA sequencing. Our method maps cellular RNA structure transcriptome-wide with temporal resolution enabling determination of transcript age-dependent RNA structural dynamics. We benchmark our approach against known RNA structures and investigate the dynamics of human 5S rRNA during ribosome biogenesis, revealing specific structural changes in 5S rRNA loops that occur over the course of several hours. Taken together, our work sheds light on the maturation and coordinated conformational changes that take place during ribosome biogenesis and provides a general strategy for surveying evolving RNA structural dynamics across the transcriptome. 

Abstract Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m⁵C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m⁵C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm⁵C- and f⁵C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m⁵C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f⁵C sites on select tRNA. Finally, we show that f⁵C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m⁵C dioxygenases and mapping oxidative m⁵C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function. 

Abstract Chemical modifications on RNA can regulate fundamental biological processes. Recent efforts have illuminated the chemical diversity of posttranscriptional (“epitranscriptomic”) modifications on eukaryotic messenger RNA and have begun to elucidate their biological roles. In this review, we discuss our current molecular understanding of epitranscriptomic RNA modifications and their effects on gene expression. In particular, we highlight the role of modifications in mediating RNA‐protein interactions, RNA structure, and RNA‐RNA base pairing, and how these macromolecular interactions control biological processes in the cell. 

Ralph Kleiner (Princeton University, USA), Claudia Höbartner (University of Würzburg, Germany) and Guifang Jia (Peking University, China) introduce the themed collection on ‘The Epitranscriptome’.