Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

FICC-Seq: A method for enzyme-specified profiling of methyl-5-uridine in cellular RNA

ABSTRACT: Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNAs, and is formed via catalytic conversion of uridines. In yeast, m5U is known to be present at position 54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to map the relevant enzymatic targets in cellular RNA. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it ubiquitously targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated demonstration of irreversible human m5U enzyme-RNA crosslinking in vivo following 5-flurouracil exposure is further intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of 5-fluorouracil-mediated cytotoxicity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE109183 | GEO | 2019/07/03

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Human TRMT2A methylates different components of the translation machinery and contributes to translation fidelity

Project description:Methyl-5-uridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme of m5U conversion at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by TRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with crosslinking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that TRMT2A interacts with proteins involved in both tRNA and rRNA biogenesis. Consistent with this finding, we observed that TRMT2A not only methylates tRNA, but also rRNA. Finally, we addressed the question of the importance of TRMT2A function by showing that its knockdown reduces translational fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards rRNA biogenesis and translational fidelity.

2023-07-04 | PXD036538 | Pride

RNA-mediated activity-based protein profiling reveals dihydrouridine modification on human mRNA

Project description:Epitranscriptomic RNA modifications can regulate RNA activity, however there remains a major gap in our understanding of the scope of mRNA chemistry present in biological systems. Here, we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy relying upon metabolic RNA labeling with the mechanism-based inhibitor 5-fluorocytidine, mRNA interactome capture, and quantitative proteomics, to investigate pyrimidine-modifying enzymes in human cells. In addition to profiling 5-methylcytidine (m5C) methyltransferase activity on mRNA, metabolic deamination of 5-fluorocytidine to 5-fluorouridine allowed us to show that 5-methyluridine (m5U) is present on human mRNA and demonstrate that its formation is primarily mediated by the tRNA-modifying enzyme TRMT2A. Further, our approach uncovered DUS3L, the mammalian homolog of the yeast tRNA-dihydrouridine synthase DUS3, as a novel 5-fluoropyrimidine-reactive protein. We investigate the mechanism of protein-RNA crosslinking, which involves the conserved catalytic Cys residue, and use genetic knockdown combined with quantitative LC-MS/MS analysis to establish that DUS3L installs dihydrouridine (DHU) on human mRNA and small RNA. Finally, we find that DUS3L is important for cell proliferation and protein translation. Taken together, our work provides a general approach for profiling RNA modifying enzyme activity in vivo, and reveals the existence of new pathways for the epitranscriptomic regulation of mRNA behavior in human cells.

2021-07-16 | PXD022645 | Pride

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Project description:While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

2024-08-08 | GSE273506 | GEO

Determination of 5-methyluridine in messenger RNA of mammals

Project description:The discovery of dynamic and reversible modifications in messenger RNA (mRNA) is opening new directions in RNA modification-mediated regulation of biological processes. Methylation is the most prevalent modification occurring in mRNA and the methylation group is mainly decorated in the adenine, cytosine, and guanine base, or in the 2’-hydroxyl group of ribose. However, methylation of the uracil base (5-methyluridine, m5U) hasn’t been discovered in mRNA of eukaryotes. In the current study, we established a method by N-cyclohexyl-N’-β-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) labelling coupled with liquid chromatography-electrospray ionization-mass spectrometry analysis (LC-ESI-MS/MS) for the sensitive determination of uridine modifications in RNA. Our results demonstrated that the detection sensitivities of uridine modifications in RNA increased up to 1543 folds upon CMCT labelling. Using the developed method, we identified the distinct existence of m5U in mRNA of various mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methylation group of m5U originated from S-Adenosyl-L-methionine (SAM). Our study expanded the list of modifications occurring in mRNA of mammals. Future work on transcriptome-wide mapping of m5U will further uncover the functional roles of m5U in mRNA of mammals.

2019-07-30 | GSE135049 | GEO

Dts-seq: a simple method of library preparation for a highly reproducible characterization of the tRNA epitranscriptome by deep sequencing

Project description:High-throughput sequencing of cellular tRNAs is severely hindered by the presence of base modifications. These modifications impair the reverse transcription (RT) enzyme and prevent a large fraction of transcripts to be converted into cDNA in conventional sequence library preparation. Recent attempts to circumvent this issue made use of enzymatic treatments to remove methyl groups (Cozen et al. 2015; Zeng et al. 2015). This approach is, however, not fully satisfactory because it can clear the way to the RT enzyme for only a fraction of all tRNAs. Another approach takes advantage of the interference of modifications with RT enzymes to detect and identify them in fragmented RNA transcripts (Hauenschild et al. 2015; Helm and Motorin 2017; Schwartz and Motorin 2017). In line with this second approach, we present results of an alternate method that is simpler and fully adapted to the direct characterization and quantification of mature tRNA transcripts. An analysis of the obtained read coverages enables to generate highly reproductible patterns of termination signals that can be used to assess the modification state of all tRNAs.

2019-08-19 | GSE135937 | GEO

Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP

Project description:The participation of transfer RNAs (tRNAs) in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes, and curation of precursor and mature tRNA sequences. This has been challenging, mainly because RNA secondary structure and nucleotide modifications, together with tRNA gene multiplicity, complicate sequencing and sequencing read mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, we further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. Our results show that approximately half of all predicted tRNA genes are transcribed in human cells. We also report predominant nucleotide modification sites, their order of introduction, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies, we present a human tRNA atlas, and determine expression levels of mature and processing intermediates of tRNAs in human cells.

2017-07-17 | GSE95683 | GEO

Crosslinked elastin-like polypeptides mediate direct cardiac differentiation of mouse embryonic and induced pluripotent stem cells without use of cardiac differentiation factors

Project description:Elastin-like polypeptides (ELPs) are promising for biomedical applications due to their unique thermos-responsive and elastic properties. ELP-based biomaterial has been produced through enzymatic crosslinking of modified ELPs. We investigated the role of elastin like polypeptide in cardiomyocyte proliferation and differentiation in mouse ES cells

2024-02-12 | GSE145623 | GEO

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Project description:Pseudouridine (ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), where is contributes ~10% of the total uridine level. However, ψ constitutes only ~0.3% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have however been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based ψ mapping technique called photo-crosslinking assisted ψ sequencing (PA-ψ-seq) and then use it to map ψ residues on not only multiple cellular RNAs but also the mRNAs and genomic RNA encoded by HIV-1. We also describe several 293T-derived cell lines in which human PUS enzymes previously reported to add ψ residues to mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were individually inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of ψ addition on cellular mRNAs to each of these three PUS enzymes, the ψ sites present on HIV-1 transcripts remained unaffected. However, loss of PUS1 function did significantly reduce HIV-1 gene expression by a presumably indirect mechanism.

2021-09-01 | GSE172136 | GEO

Architecture of the yeast Elongator complex

Project description:The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub-complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2 and Elp3 subunits form a two-lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.

2016-12-20 | PXD005251 | Pride

Growth Optimized Aminoacyl-tRNA Synthetase Levels Prevent Maximal tRNA Charging

Project description:Summary Aminoacyl-tRNA synthetases (aaRSs) serve a dual role in charging tRNAs. Their enzymatic activities both provide protein synthesis flux and reduce uncharged tRNA levels. Although uncharged tRNAs can negatively impact bacterial growth, substantial concentrations of tRNAs remain deacylated even under nutrient-rich conditions. Here we show that tRNA charging in Bacillus subtilis is not maximized due to optimization of aaRS production during rapid growth, which prioritizes demands in protein synthesis over charging levels. The presence of uncharged tRNAs is alleviated by precisely tuned translation kinetics and the stringent response, both insensitive to aaRS overproduction but sharply responsive to underproduction, allowing for just-enough aaRS production atop a “fitness cliff”. Notably, we find that the stringent response mitigates fitness defects at all aaRS underproduction levels even without external starvation. Thus, adherence to minimal, flux-satisfying protein production drives limited tRNA charging and provides a basis for the sensitivity and setpoints of an integrated growth-control network.

2020-07-20 | GSE141389 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data