Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

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), RNA-protein enrichment, and quantitative proteomics. We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and use quantitative nucleoside LC-MS to show that ALKBH1 is the major hm5C and f5C-forming enzyme in RNA, including upon polyadenylated RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-based iCLIP analysis to show that ALKBH1 oxidizes m5C in a variety of tRNA anticodon stem loops (ASL), as well as on mRNA and lncRNA, and analyze its substrate specificity using in vitro enzymatic assays. Finally, we apply pyridine borane-mediated sequencing to identify f5C sites in tRNA ASLs. 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Emilia Arguello

LAB HEAD: Ralph E. Kleiner

PROVIDER: PXD029955 | Pride | 2022-07-16

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	04252019_Kleiner_EA_595_EA_iii_76A_20190501190138.raw	Raw
	04252019_Kleiner_EA_595_EA_iii_76C_20190502060403.raw	Raw
	05062019_Kleiner_EA_605_EA_iii_76A_2.raw	Raw
	05062019_Kleiner_EA_605_EA_iii_76C_2.raw	Raw
	EA-iii-76_3_rep_5EC_spectral_counts.xlsx	Xlsx

Items per page:

1 - 5 of 8

Similar Datasets

RBDmap - Comprehensive Identification of RNA-Binding Domains in Human Cells

Project description:The RNA interactomes of HeLa and HEK293 cells jointly comprise 1106 RNA-binding proteins (RBPs), with almost half of these lacking well-defined RNA-binding domains (RBDs), suggesting the existence of numerous unknown RNA-binding architectures. Here, we report on “RBDmap”, a new method built on interactome capture, to comprehensively identify the RBDs of native RBPs in HeLa cells. Making use of in vivo UV-crosslinking of RBPs to polyadenylated RNAs, capture on oligo(dT) magnetic beads, sequential proteolytic digestion and LC-MS/MS combined with a sophisticated scoring algorithm, RBDmap “re-discoveres” many known RNA-binding sites (e.g. RRM, KH) of numerous well characterized RBPs and suggests novel RBDs.

2016-08-08 | PXD000883 | Pride

Serial interactome capture of the human cell nucleus

Project description:Novel RNA-guided cellular functions are paralleled by an increasing number of RNA binding proteins (RBPs). We present “serial interactome capture” (serIC), a multiple purification procedure of UV-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with maximal specificity. We apply serIC to nuclei of proliferating K562 cells to obtain the first human nuclear interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including fewer factors without known RNA binding domains that are in better agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signaling and double strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs. The nuclear interactome presented here is a stepping-stone towards deciphering of the functional RNA-protein network in the mammalian nucleus.

2016-03-31 | PXD003664 | Pride

Single-cell RNA-seq on Drosophila melanogaster wild type third instar eye discs

Project description:Wandering third instar larvae were dissected where the eye disc was separated from the antenna and brain. The tissues were dissociated into single cells and captured using Drop-seq. Single cell libraries were then generated from each cell and finally sequenced.

2018-10-29 | E-MTAB-7195 | biostudies-arrayexpress

mRNA binding - Insights into RNA biology from an atlas of mammalian mRNA-binding proteins

Project description:RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.

2015-09-11 | PXD002880 | Pride

RNA binding proteins modification by drought stress

Project description:RNA-binding proteins are increasingly recognized as regulatory component of post-transcriptional gene expression. RBPs interact with mRNAs via RNA-binding domains and these interactions affect RNA availability for translation, RNA stability and turn-over thus affecting both RNA and protein expression essential for developmental and stimulus specific responses. Here we investigate the effect of severe drought stress on the RNA-binding proteome to gain insights into the mechanisms that govern drought stress responses at the systems level

2020-05-27 | PXD016883 | Pride

Enrichment of processed pseudogene transcripts in L1-ribonucleoprotein particles

Project description:We explored the RNA binding properties of LINE-1 ORF1p, both free and in the L1 RNP, using a recently developed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation technique (PAR-CLIP) to comprehensively identify ORF1p binding sites in the transcriptome of human cells (HEK293T). Our results show that ORF1p binds to a wide range of cellular mRNAs, with an enrichment for binding at the 3’ UTR. Our data also show that ORF1p binds very strongly with retrotransposable RNA, i.e., L1, Alu and SVA. PAR-CLIP analysis of L1 RNPs and free ORF1p RNA binding profiles, comparison to HuR RNA binding profile

2013-05-21 | E-GEOD-43801 | biostudies-arrayexpress

Transcriptome-wide Identification of Single-stranded RNA Binding Proteins

Project description:sinle-stranded RNA binding proteins were identified using DIA MS with the asist of RNA seconderay structure specific probe

2023-05-10 | PXD038757 | Pride

RNA m5C Methylation in breast cancer using MeRIP-Seq

Project description:RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody. Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700). m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA. This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.

2014-02-20 | E-GEOD-53370 | biostudies-arrayexpress

Epigenetic comparison of human DNA in specific human tissues to human DNA in transgenic mouse tissues

Project description:This project aims to study human repetitive elements on human chromosome 21 (HsChr21) in a heterologous mouse environment (Tc1 mouse). The in vivo, HsChr21-wide enrichment of active histone modified regions (H3K4me3) and transcription factor binding sites was profiled by ChIP-seq in Human and Tc1 mouse tissue in order to determine the unbiased regulation of the human genome.

2013-03-21 | E-MTAB-1104 | biostudies-arrayexpress

Epitranscriptomic cytidine methylation of the Hepatitis B viral RNA encapsidation signal ensure the reverse transcription of viral RNA [PA-m5C-seq]

Project description:Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of both cellular and viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation and splicing of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remain elusive. Here, we report that the RNA of hepatitis B virus (HBV) is enriched with a high level of m5C, mediated mainly through the cellular methyltransferase NSUN2. Intrigingly, the most prominent cluster of NSUN2-deposited m5C is found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription. Loss of m5C from HBV RNA due to depletion of NSUN2 resulted in a modest decrease in viral capsid protein (HBc) translation, yet this is accompaneied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a translation decrease and block of reverse transcription. Furthermore, pharmacological disruption of m5C deposition with a nucleoside analogue led to a significant decrease in HBV replication. Thus, our data indicates m5C methylations is a critical enhancer of the epsilon element in HBV reverse transcription. Our study suggests the theraputic potential of targeting the m5C methyltransfer process on the HBV 5’epsilon as an alternative antiviral stratagy.

2024-05-20 | GSE250106 | GEO