Introduction to 'The Epitranscriptome'.

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

Sequence- and Structure-Specific tRNA Dihydrouridylation by hDUS2.

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.

The impact of epitranscriptomic modifications on liver disease.

RNA modifications have emerged as important mechanisms of gene regulation. Developmental, metabolic, and cell cycle regulatory processes are all affected by epitranscriptomic modifications, which control gene expression in a dynamic manner. The hepatic tissue is highly metabolically active and has an impressive ability to regenerate after injury. Cell proliferation, differentiation, and metabolism, which are all essential to the liver response to injury and regeneration, are regulated via RNA modification. Two such modifications, N6-methyladenosine (m6A)and 5-methylcytosine (m5C), have been identified as prognostic disease markers and potential therapeutic targets for liver diseases. Here, we describe progress in understanding the role of RNA modifications in liver biology and disease and discuss specific areas where unexpected results could lead to improved future understanding.

Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

Cellular nucleic acids are subject to assault by endogenous and exogenous agents that can perturb the flow of genetic information. Oxidative stress leads to the accumulation of 8-oxoguanine (8OG) in DNA and RNA. 8OG lesions on mRNA negatively impact translation, but their effect on global RNA-protein interactions is largely unknown. Here, we apply an RNA chemical proteomics approach to investigate the effect of 8OG on RNA-protein binding. We find proteins that bind preferentially to 8OG-modified RNA, including IGF2BP1-3 and hnRNPD, and proteins that are repelled by 8OG such as RBM4. We characterize these interactions using biochemical and biophysical assays to quantify the effect of 8OG on binding and show that a single 8OG abolishes the binding of RBM4 to its preferred CGG-containing substrate. Taken together, our work establishes the molecular consequences of 8OG on cellular RNA-protein binding and provides a framework for interrogating the role of RNA oxidation in biological systems.

Cell type-specific translational regulation by human DUS enzymes.

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.

A minimal sequence motif drives selective tRNA dihydrouridylation by hDUS2.

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 and in vitro dihydrouridylation 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.

Chemoproteomic Approaches to Studying RNA Modification-Associated Proteins.

The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for N6-methyladenosine (m6A) and N1-methyladenosine (m1A) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both m6A and m1A to mediate transcript destabilization. Furthermore, m6A also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.

Chemical Approaches To Investigate Post-transcriptional RNA Regulation.

RNA plays a central role in biological processes, and its activity is regulated by a host of diverse chemical and biochemical mechanisms including post-transcriptional modification and interactions with RNA-binding proteins. Here, we describe our efforts to illuminate RNA biology through the application of chemical tools, focusing on post-transcriptional regulatory mechanisms. We describe the development of an activity-based protein profiling approach for discovery and characterization of RNA-modifying enzymes. Next, we highlight novel approaches for RNA imaging based upon metabolic labeling with modified nucleosides and engineering of the nucleotide salvage pathway. Finally, we discuss profiling RNA-protein interactions using small molecule-dependent RNA editing and synthetic photo-cross-linkable oligonucleotide probes. Our work provides enabling technologies for deciphering the complexity of RNA and its diverse functions in biology.

Inhibition of nucleolar transcription by oxaliplatin involves ATM/ATR kinase signaling.

Platinum (Pt) compounds are an important class of anti-cancer therapeutics, but outstanding questions remain regarding their mechanism of action. Here, we demonstrate that oxaliplatin, a Pt drug used to treat colorectal cancer, inhibits rRNA transcription through ATM and ATR signaling, and induces DNA damage and nucleolar disruption. We show that oxaliplatin causes nucleolar accumulation of the nucleolar DNA damage response proteins (n-DDR) NBS1 and TOPBP1; however transcriptional inhibition does not depend upon NBS1 or TOPBP1, nor does oxaliplatin induce substantial amounts of nucleolar DNA damage, distinguishing the nucleolar response from previously characterized n-DDR pathways. Taken together, our work indicates that oxaliplatin induces a distinct ATM and ATR signaling pathway that functions to inhibit Pol I transcription in the absence of direct nucleolar DNA damage, demonstrating how nucleolar stress and transcriptional silencing can be linked to DNA damage signaling and highlighting an important mechanism of Pt drug cytotoxicity.

Profiling dynamic RNA-protein interactions using small-molecule-induced RNA editing.

RNA-binding proteins (RBPs) play an important role in biology, and characterizing dynamic RNA-protein interactions is essential for understanding RBP function. In this study, we developed targets of RBPs identified by editing induced through dimerization (TRIBE-ID), a facile strategy for quantifying state-specific RNA-protein interactions upon rapamycin-mediated chemically induced dimerization and RNA editing. We performed TRIBE-ID with G3BP1 and YBX1 to study RNA-protein interactions during normal conditions and upon oxidative stress-induced biomolecular condensate formation. We quantified editing kinetics to infer interaction persistence and show that stress granule formation strengthens pre-existing RNA-protein interactions and induces new RNA-protein binding events. Furthermore, we demonstrate that G3BP1 stabilizes its targets under normal and oxidative stress conditions independent of stress granule formation. Finally, we apply our method to characterize small-molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile dynamic RNA-protein interactions in cellular contexts with temporal control.

Global Discovery of Covalent Modulators of Ribonucleoprotein Granules.

Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.

Oxaliplatin Inhibits RNA Polymerase I via DNA Damage Signaling Targeted to the Nucleolus.

Platinum (Pt) compounds are an important class of anti-cancer therapeutics, but outstanding questions remain regarding their mode of action. In particular, emerging evidence indicates that oxaliplatin, a Pt drug used to treat colorectal cancer, kills cells by inducing ribosome biogenesis stress rather than through DNA damage generation, but the underlying mechanism is unknown. Here, we demonstrate that oxaliplatin-induced ribosomal RNA (rRNA) transcriptional silencing and nucleolar stress occur downstream of DNA damage signaling involving ATM and ATR. We show that NBS1 and TOPBP1, two proteins involved in the nucleolar DNA damage response (n-DDR), are recruited to nucleoli upon oxaliplatin treatment. However, we find that rRNA transcriptional inhibition by oxaliplatin does not depend upon NBS1 or TOPBP1, nor does oxaliplatin induce substantial amounts of nucleolar DNA damage, distinguishing it from previously characterized n-DDR pathways. Taken together, our work indicates that oxaliplatin induces a distinct DDR signaling pathway that functions in trans to inhibit Pol I transcription in the nucleolus, demonstrating how nucleolar stress can be linked to DNA damage signaling and highlighting an important mechanism of Pt drug cytotoxicity.

Tracking chromatin state changes using nanoscale photo-proximity labelling.

Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.

Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides.

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems; however, approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2. In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Furthermore, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases.

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.

Chemical Method to Sequence 5-Formylcytosine on RNA.

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method for sequencing 5-formylcytosine (f5C) modifications on RNA based on the selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and polymerase chain reaction amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

Interrogating the transcriptome with metabolically incorporated ribonucleosides.

RNA is a central player in biological processes, but there remain major gaps in our understanding of transcriptomic processes and the underlying biochemical mechanisms regulating RNA in cells. A powerful strategy to facilitate molecular analysis of cellular RNA is the metabolic incorporation of chemical probes. In this review, we discuss current approaches for RNA metabolic labeling with modified ribonucleosides and their integration with Next-Generation Sequencing, mass spectrometry-based proteomics, and fluorescence microscopy in order to interrogate RNA behavior in its native context.

Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation.

Epitranscriptomic RNA modifications can regulate RNA activity; however, there remains a major gap in our understanding of the RNA chemistry present in biological systems. Here we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy that relies on metabolic RNA labeling, mRNA interactome capture and quantitative proteomics, to investigate RNA-modifying enzymes in human cells. RNABPP with 5-fluoropyrimidines allowed us to profile 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferases. Further, we uncover a new mechanism-based crosslink between 5-fluorouridine (5-FUrd)-modified RNA and the dihydrouridine synthase (DUS) homolog DUS3L. We investigate the mechanism of crosslinking and use quantitative nucleoside liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and 5-FUrd-based crosslinking and immunoprecipitation (CLIP) sequencing to map DUS3L-dependent dihydrouridine (DHU) modifications across the transcriptome. Finally, we show that DUS3L-knockout (KO) cells have compromised protein translation rates and impaired cellular proliferation. Taken together, our work provides a general approach for profiling RNA-modifying enzyme activity in living cells and reveals new pathways for epitranscriptomic RNA regulation.

A neural m6A/Ythdf pathway is required for learning and memory in Drosophila.

Epitranscriptomic modifications can impact behavior. Here, we used Drosophila melanogaster to study N6-methyladenosine (m6A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m6A binders. Assays of short term memory in m6A mutants reveal neural-autonomous requirements of m6A writers working via Ythdf, but not Ythdc1. Furthermore, m6A/Ythdf operate specifically via the mushroom body, the center for associative learning. We map m6A from wild-type and Mettl3 mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m6A is preferentially deposited on genes with low translational efficiency and that m6A does not affect RNA stability. Nevertheless, functional tests indicate a role for m6A/Ythdf in translational activation. Altogether, our molecular genetic analyses and tissue-specific m6A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.