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RNA-mediated activity-based protein profiling reveals the substrates of a human dihydrouridine synthase

ABSTRACT: RNA-mediated activity-based protein profiling reveals the substrates of a human dihydrouridine synthase

PROVIDER: PRJNA733999 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14693254.fastq.gz	Fastqsanger.gz
	SRR14693255.fastq.gz	Fastqsanger.gz
	SRR14693256.fastq.gz	Fastqsanger.gz
	SRR14693257.fastq.gz	Fastqsanger.gz
	SRR14693258.fastq.gz	Fastqsanger.gz

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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

RNA-mediated activity-based protein profiling reveals the substrates of a human dihydrouridine synthase

Project description:We adapted the iCLIP method to map DUS3L-dependent DHU sites in a transcriptome-wide manner by treating the cells with 5-FUrd.

2021-06-01 | GSE175825 | GEO

Exploring a Novel Class of Flavoenzymes: Identification and Biochemical Characterization of Ribosomal RNA Dihydrouridine Synthase

Project description:New Ribosomal RNA Dihydrouridine Synthase in E.coli

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Transcriptome-wide mapping of dihydrouridine sites with D-seq

Project description:We report the development of D-Seq, a sequencing based technique for identification of dihydrouridine (D) modifications within RNA. We performed D-Seq on S. cerevisiae and find the first evidence for dihydrouridylation of endogenous mRNAs.

2022-04-16 | GSE175549 | GEO

Fruquintinib DDI Study With P-gp and BCRP Substrates

Project description:Fruquintinib DDI Study with P-gp and BCRP Substrates

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O-GlcNAc transferase recognizes protein substrates using an asparagine ladder in the TPR superhelix

Project description:Abstract: The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we have developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously as-sess OGT activity across >6000 human proteins. We used this assay to examine the contribution of a conserved asparagine ladder within the lumen of OGT’s superhelical tetratri-copeptide repeat (TPR) domain to substrate selection. When these residues were mutated, OGT retained full activity against short peptides, but showed low to no activity against most of the OGT substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes a majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain. This series contains microarray data both comparing the new chemoenzymatic method to antibody-based detection as well as comparing arrays treated with wild-type OGT, 5N5A mutant OGT, or controls not treated with enzyme. Note: all CTD-stained arrays or control array raw files are contained in GSE107911_RAW.tar

2018-02-26 | GSE107911 | GEO

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases.

Project description:The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNA(Phe) and tRNA(Trp) show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids ("binding signatures") together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal "recognition" domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.

| S-EPMC4434734 | biostudies-literature

Structure of dihydrouridine synthase C (DusC) from Escherichia coli.

Project description:Dihydrouridine (D) is one of the most widely conserved tRNA modifications. Dihydrouridine synthase (Dus) is responsible for introducing D modifications into RNA by the reduction of uridine. Recently, a unique substrate-recognition mechanism using a small adapter molecule has been proposed for Thermus thermophilus Dus (TthDusC). To acquire insight regarding its substrate-recognition mechanism, the crystal structure of DusC from Escherichia coli (EcoDusC) was determined at 2.1 Å resolution. EcoDusC was shown to be composed of two domains: an N-terminal catalytic domain and a C-terminal tRNA-binding domain. An L-shaped electron density surrounded by highly conserved residues was found in the active site, as observed for TthDus. Structure comparison with TthDus indicated that the N-terminal region has a similar structure, whereas the C-terminal domain has marked differences in its relative orientation to the N-terminal domain as well as in its own structure. These observations suggested that Dus proteins adopt a common substrate-recognition mechanism using an adapter molecule, whereas the manner of tRNA binding is diverse.

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From bacterial to human dihydrouridine synthase: automated structure determination.

Project description:The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1-340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr_rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain-domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

| S-EPMC4498606 | biostudies-literature

Transcriptome-wide mapping of dihydrouridine sites with D-seq

Project description:Transcriptome-wide mapping of dihydrouridine sites with D-seq

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