PXD022645-1

PXD022645 is an original dataset announced via ProteomeXchange.

Title	RNA-mediated activity-based protein profiling reveals dihydrouridine modification on human mRNA
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.
HostingRepository	PRIDE
AnnounceDate	2021-07-16
AnnouncementXML	Submission_2021-07-15_23:21:18.566.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Thao Nguyen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2020-11-20 02:58:11	ID requested
⏵ 1	2021-07-15 23:21:18	announced

Dataset with its publication pending

submitter keyword: RNA modification, metabolic RNA labeling, LC-MS/MS

Martin Wuhr
contact affiliation	Department of Chemistry, Princeton University, Princeton, NJ 08544, USA Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08540, USA
contact email	wuhr@princeton.edu
lab head
Thao Nguyen
contact affiliation	Wuhr Lab - Princeton University
contact email	tn3@princeton.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD022645
     1. Label: PRIDE project
     2. Name: RNA-mediated activity-based protein profiling reveals dihydrouridine modification on human mRNA