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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

ABSTRACT: Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

PROVIDER: PRJNA837404 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16992326.fastq.gz	Fastqsanger.gz
	SRR16992327.fastq.gz	Fastqsanger.gz
	SRR19175036.fastq.gz	Fastqsanger.gz
	SRR19175037.fastq.gz	Fastqsanger.gz
	SRR19175038.fastq.gz	Fastqsanger.gz

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

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

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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.

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 [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.

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Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

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

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