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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases [II]

ABSTRACT: Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases [II]

PROVIDER: PRJNA837406 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19175036.fastq.gz	Fastqsanger.gz
	SRR19175037.fastq.gz	Fastqsanger.gz
	SRR19175038.fastq.gz	Fastqsanger.gz
	SRR19175039.fastq.gz	Fastqsanger.gz
	SRR19175040.fastq.gz	Fastqsanger.gz

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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Project description:Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases [I]

Project description:Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases [I]

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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Project description: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.

2022-07-16 | PXD029955 | Pride

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases [II]

Project description:We adapted the Aldehyde reactive probe (ARP; O-(biotinylcarbazoylmethyl) hydroxylamine) pulldown method to map ALKBH1 dependent targets in a transcriptome-wide manner.

2022-06-28 | GSE202814 | GEO

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases.

Project description: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.

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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-06-28 | GSE202815 | GEO

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases [I]

Project description:We adapted the iCLIP method to map ALKBH1L modification sites in a transcriptome-wide manner by treating the cells with 5-EC.

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Project description:Proteomics has revealed that the ~20,000 human genes engender a far greater number of proteins, or proteoforms, that are diversified in large part by post-translational modifications (PTMs). How such PTMs affect protein structure and function is an active area of research but remains technically challenging to assess on a proteome-wide scale. Here, we describe a chemical proteomic method to quantitatively relate serine/threonine phosphorylation to changes in the reactivity of cysteine residues, a parameter that can affect the potential for cysteines to be post-translationally modified or engaged by covalent drugs. Leveraging the extensive high-stoichiometry phosphorylation occurring in mitotic cells, we discover numerous cysteines that exhibit phosphorylation-dependent changes in reactivity on diverse proteins enriched in cell cycle regulatory pathways. The discovery of bidirectional changes in cysteine reactivity often occurring in proximity to serine/threonine phosphorylation events points to the broad impact of phosphorylation on the chemical reactivity of proteins and the future potential to create small-molecule probes that differentially target PTM-modified proteoforms.

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