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

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

ORGANISM(S): Homo sapiens

PROVIDER: GSE189261 | GEO | 2022/06/28

REPOSITORIES: GEO

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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

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

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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). 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.

| S-EPMC9296451 | biostudies-literature

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

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

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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 [I]

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

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Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition

Project description:We adapted the iCLIP method to zebrafish embryos to investigate activities of Hnrnpa1 during the maternal-to-zygotic transition

2017-03-24 | GSE76212 | GEO

Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3M-bM-^@M-^Y UTRs

Project description:The RNA helicase UPF1 is best known for its key function in mRNA nonsense-mediated mRNA decay (NMD), but has also been implicated in additional mRNA turnover mechanisms, telomere homeostasis, and DNA replication. In NMD, UPF1 recruitment to target mRNAs is thought to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3M-bM-^@M-^Y untranslated region (UTR) of mRNAs has also been reported. To map UPF1 binding sites transcriptome-wide, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation by puromycin. We found a strong association of UPF1 with 3M-bM-^@M-^Y UTRs in undisturbed, translationally active cells and a significant increase in UPF1 binding to coding sequence (CDS) after translation inhibition. These results indicate that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This evidence for translation-independent UPF1-RNA interaction, which is corroborated by RNA immunoprecipitations experiments and by our observation that UPF1 also crosslinks to long non-coding RNAs, suggests that the decision to trigger NMD occurs after association of UPF1 with the mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Examination of Upf1 binding preferences via iCLIP in untreated HeLa cells and HeLa cells, where translation is blocked by puromycin treatment in vivo crosslinking and immunoprecipitation strategy (iCLIP)

2013-06-20 | E-GEOD-47976 | biostudies-arrayexpress

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