skip to main content

Title: 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.

; ; ; ; ;
Award ID(s):
Publication Date:
Journal Name:
Nature Communications
Nature Publishing Group
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract Background

    Emerging RNA viruses that target the central nervous system (CNS) lead to cognitive sequelae in survivors. Studies in humans and mice infected with West Nile virus (WNV), a re-emerging RNA virus associated with learning and memory deficits, revealed microglial-mediated synapse elimination within the hippocampus. Moreover, CNS-resident memory T (TRM) cells activate microglia, limiting synapse recovery and inducing spatial learning defects in WNV-recovered mice. The signals involved in T cell-microglia interactions are unknown.


    Here, we examined immune cells within the murine WNV-recovered forebrain using single-cell RNA sequencing to identify putative ligand-receptor pairs involved in intercellular communication between T cells and microglia. Clustering and differential gene analyses were followed by protein validation and genetic and antibody-based approaches utilizing an established murine model of WNV recovery in which microglia and complement promote ongoing hippocampal synaptic loss.


    Profiling of host transcriptome immune cells at 25 days post-infection in mice revealed a shift in forebrain homeostatic microglia to activated subpopulations with transcriptional signatures that have previously been observed in studies of neurodegenerative diseases. Importantly, CXCL16/CXCR6, a chemokine signaling pathway involved in TRM cell biology, was identified as critically regulating CXCR6 expressing CD8+TRM cell numbers within the WNV-recovered forebrain. We demonstrate that CXCL16 is highlymore »expressed by all myeloid cells, and its unique receptor, CXCR6, is highly expressed on all CD8+T cells. Using genetic and pharmacological approaches, we demonstrate that CXCL16/CXCR6 not only is required for the maintenance of WNV-specific CD8 TRM cells in the post-infectious CNS, but also contributes to their expression of TRM cell markers. Moreover, CXCR6+CD8+T cells are required for glial activation and ongoing synapse elimination.


    We provide a comprehensive assessment of the role of CXCL16/CXCR6 as an interaction link between microglia and CD8+T cells that maintains forebrain TRM cells, microglial and astrocyte activation, and ongoing synapse elimination in virally recovered animals. We also show that therapeutic targeting of CXCL16 in mice during recovery may reduce CNS CD8+TRM cells.

    « less
  2. Abstract Ribonucleotides within the various RNA molecules in eukaryotes are marked with more than 160 distinct covalent chemical modifications. These modifications include those that occur internally in messenger RNA (mRNA) molecules such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), as well as those that occur at the ends of the modified RNAs like the non-canonical 5′ end nicotinamide adenine dinucleotide (NAD+) cap modification of specific mRNAs. Recent findings have revealed that covalent RNA modifications can impact the secondary structure, translatability, functionality, stability and degradation of the RNA molecules in which they are included. Many of these covalent RNA additions have also been found to be dynamically added and removed through writer and eraser complexes, respectively, providing a new layer of epitranscriptome-mediated post-transcriptional regulation that regulates RNA quality and quantity in eukaryotic transcriptomes. Thus, it is not surprising that the regulation of RNA fate mediated by these epitranscriptomic marks has been demonstrated to have widespread effects on plant development and the responses of these organisms to abiotic and biotic stresses. In this review, we highlight recent progress focused on the study of the dynamic nature of these epitranscriptome marks and their roles in post-transcriptional regulation during plant development and response to environmentalmore »cues, with an emphasis on the mRNA modifications of non-canonical 5′ end NAD+ capping, m6A and several other internal RNA modifications.« less
  3. Abstract Background

    Direct-sequencing technologies, such as Oxford Nanopore’s, are delivering long RNA reads with great efficacy and convenience. These technologies afford an ability to detect post-transcriptional modifications at a single-molecule resolution, promising new insights into the functional roles of RNA. However, realizing this potential requires new tools to analyze and explore this type of data.


    Here, we present Sequoia, a visual analytics tool that allows users to interactively explore nanopore sequences. Sequoia combines a Python-based backend with a multi-view visualization interface, enabling users to import raw nanopore sequencing data in a Fast5 format, cluster sequences based on electric-current similarities, and drill-down onto signals to identify properties of interest. We demonstrate the application of Sequoia by generating and analyzing ~ 500k reads from direct RNA sequencing data of human HeLa cell line. We focus on comparing signal features from m6A and m5C RNA modifications as the first step towards building automated classifiers. We show how, through iterative visual exploration and tuning of dimensionality reduction parameters, we can separate modified RNA sequences from their unmodified counterparts. We also document new, qualitative signal signatures that characterize these modifications from otherwise normal RNA bases, which we were able to discover from the visualization.


    Sequoia’s interactive features complementmore »existing computational approaches in nanopore-based RNA workflows. The insights gleaned through visual analysis should help users in developing rationales, hypotheses, and insights into the dynamic nature of RNA. Sequoia is available at

    « less
  4. Dos Santos, P.C. (Ed.)
    Iron-Sulfur (Fe-S) clusters function as core prosthetic groups known to modulate the activity of metalloenzymes, act as trafficking vehicles for biological iron and sulfur, and participate in several intersecting metabolic pathways. The formation of these clusters is initiated by a class of enzymes called cysteine desulfurases, whose primary function is to shuttle sulfur from the amino acid l-cysteine to a variety of sulfur transfer proteins involved in Fe-S cluster synthesis as well as in the synthesis of other thiocofactors. Thus, sulfur and Fe-S cluster metabolism are connected through shared enzyme intermediates, and defects in their associated pathways cause a myriad of pleiotropic phenotypes, which are difficult to dissect. Post-transcriptionally modified transfer RNA (tRNA) represents a large class of analytes whose synthesis often requires the coordinated participation of sulfur transfer and Fe-S enzymes. Therefore, these molecules can be used as biologically relevant readouts for cellular Fe and S status. Methods employing LC-MS technology provide a valuable experimental tool to determine the relative levels of tRNA modification in biological samples and, consequently, to assess genetic, nutritional, and environmental factors modulating reactions dependent on Fe-S clusters. Herein, we describe a robust method for extracting RNA and analytically evaluating the degree of Fe-S-dependent andmore »-independent tRNA modifications via an LC-MS platform.« less
  5. Shank, Elizabeth Anne (Ed.)
    ABSTRACT Posttranscriptional modifications to tRNA are critical elements for the folding and functionality of these adaptor molecules. Sulfur modifications in tRNA are installed by specialized enzymes that act on cognate tRNA substrates at specific locations. Most studied organisms contain a general cysteine desulfurase to mobilize sulfur for the synthesis of S-tRNA and other thio-cofactors. Bacillus subtilis and other Gram-positive bacteria encode multiple cysteine desulfurases that partner with specific sulfur acceptors in the biosynthesis of thio-cofactors. This metabolic layout suggests an alternate mode of regulation in these biosynthetic pathways. In this study, tRNA modifications were exploited as a readout for the functionality of pathways involving cysteine desulfurases. These analyses showed that the relative abundance of 2-thiouridine-modified tRNA (s 2 U) responds to sulfur availability in the growth medium in a dose-dependent manner. This study found that low sulfur concentrations lead to decreased levels of the s 2 U cysteine desulfurase YrvO and thiouridylase MnmA, without altering the levels of other cysteine desulfurases, SufS, NifS, and NifZ. Analysis of pathway metabolites that depend on the activity of cysteine desulfurases indicates that sulfur nutrient availability specifically impacts s 2 U accumulation while having no effect on the levels of other S-modified tRNA ormore »activity levels of Fe-S enzymes. Collectively, these results support a model in which s 2 U tRNA serves as a marker for sulfur availability in B. subtilis . IMPORTANCE The 2-thiouridine (s 2 U) tRNA modification is found ubiquitously across all domains of life. YrvO and MnmA, the enzymes involved in this modification, are essential in B. subtilis , confirming the well-established role of s 2 U in maintaining translational efficiency and, consequently, cellular viability. Herein, we show that in the model Gram-positive organism Bacillus subtilis , the levels of s 2 U are responsive to sulfur availability. Downregulation of the s 2 U biosynthetic components leads to lower s 2 U levels, which may serve as a signal for the slowing of the translational apparatus during cellular nutrient insufficiency. Our findings provide the basis for the identification of a potential bacterial mode of regulation during S-metabolite depletion that may use s 2 U as a marker of suboptimal metabolic status.« less