Single-Base Resolution Mapping Reveals Distinct 5-Formylcytidine in <i>Saccharomyces cerevisiae</i> mRNAs

Wang, Ya-Fen; Chen, Zonggui; Zhang, Xiong; Weng, Xiaocheng; Deng, Jikai; Yang, Wei; Wu, Fan; Han, Shaoqing; Xia, Chao; Zhou, Yu; Chen, Yu; Zhou, Xiang

doi:10.1021/acschembio.1c00633

Cited by 19 publications

(28 citation statements)

References 39 publications

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“…Since LC-MS analysis of bulk RNA digests does not provide information on the modification site and antisense pulldowns could still contain trace amounts of non-targeted tRNA species, we decided to pursue a complementary sequencing approach to more rigorously characterize tRNA f 5 C sites. In particular, Song and co-workers demonstrated that 5fC in DNA can be converted to dihydrouracil by pyridine borane treatment, resulting in C-to-T mutations upon PCR and sequencing 54 , and while our manuscript was in this review, this method was adapted to identify RNA f 5 C sites in yeast 55 . Similarly, we previously reported Mal-Seq 56 , which uses malononitrile to detect f 5 C in RNA through C-to-T mutations, although with lower sensitivity than the pyridine borane procedure.…”

Section: Resultsmentioning

confidence: 99%

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Arguello

Sun

et al. 2022

Nat Commun

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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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Section: Resultsmentioning

confidence: 99%

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Arguello

Sun

et al. 2022

Nat Commun

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“…Unfortunately, f 5 C behaved equally to cytosine in bisulfite sequencing 56 and similarly to ca 5 C and ac 4 C in protonation-dependent hydride reduction based sequencings. 18,27,29 Besides, its low abundance in transcript RNA (10 -6 f 5 C/G) makes it challenging to efficiently employ antibody-based immunoprecipitation that normally worked well with other commonly existing modifications. To overcome such technical bottleneck, we consider that the hydrogen bond donor and acceptor sites of paC have changed significantly compared with cytosine (Figure S26), and it may no longer pair with guanine during the reverse transcription process, resulting in distinct changes in the reading signal and hence an exact indication of the formyl modification sites.…”

Section: Results and Disscusionsmentioning

confidence: 99%

“…Kleiner, Meier, Minczuk, and Zhou simultaneous disclosed two distinct chemical reduction and alkylation-based sequencing of f 5 C in RNA. 18,[27][28][29] The mostly used borohydride sequencing relied on the high polarity and electron deficiency of C5=C6 double bond conjugated with the electron-withdrawing formyl group, which was reduced under acidic conditions (pH 1 or 5.2) to afford the dihydrouridine DHU. 18,27,29 A subsequent PCR reaction enabled the conversion of DHU to thymine and thus uncovered the f5C modification in the following sequencing process.…”

Section: Introductionmentioning

confidence: 99%

“…18,[27][28][29] The mostly used borohydride sequencing relied on the high polarity and electron deficiency of C5=C6 double bond conjugated with the electron-withdrawing formyl group, which was reduced under acidic conditions (pH 1 or 5.2) to afford the dihydrouridine DHU. 18,27,29 A subsequent PCR reaction enabled the conversion of DHU to thymine and thus uncovered the f5C modification in the following sequencing process. While this method has produced signals directly at the f 5 C modification sites, it could not intrinsically differentiate it with other electron-deficient cytidines.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Photo-triggered Wittig-Cyclization for Detecting and Sequencing 5-Formylcytidine in RNA

Cheng

Jin

Huang

et al. 2022

Preprint

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The simultaneous detection of a specific post-transcriptional modification that decorates the natural RNA can provide comprehensive information to better understand cellular and molecular functions of this variation. 5-Formylcytidine f5C is one of the natural nucleotides that was discovered in the tRNA of many species during the oxidative demethylation of 5-methylcytidine m5C. Despite the evident importance of f5C in playing an important role in gene expression regulation, little is known about the exact amount and positions of this modified cytidine in RNAs, as well as its exact functions. To capture this type of information, we performed a site-specific dual functionalization of f5C with semi-stabilized ylide cyanomethylene triphenylphosphorane that can simultaneously seizure the C5-formyl and N4-amino groups in the cytidine residue under light irradiation. The photochemical labelling with f5C-containing oligonucleotides imparts a high selectivity towards f5C and allows distinction from structurally similar 5-formyluridine f5U. We implemented a detection strategy of f5C modification in RNA based on the fluorescence signal of the cyclization product 4,5-pyridin-2-amine-cytidine paC, which exhibited a relatively high fluorescence quantum yield. The results could clearly identify f5C with a limit of detection LOD at 0.58 nM. The chemical labelling was capable of altering the hydrogen bonding activity of the parent cytidine and enabled the modulation of the reverse transcription signature of f5C in sequencing profile. We showed that using this Pan-Seq strategy, f5C can be detected from tRNA segments with a single-base resolution. Taken together, this approach represents a sensitive, robust, antibody-free and applicable detection ad sequencing to f5C RNA.

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“…Some studies provide evidence that they might affect RNA stability and translation, as demonstrated for 5hmrC in Drosophila [ 23 ]. 5carC has been recently discovered in mammalian RNA [ 6 ], but there is still little knowledge about its prevalence and functions compared with those for 5hmrC or 5frC [ 20 , 22 , 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea

et al. 2022

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The formation of three oxidative DNA 5-methylcytosine (5mC) modifications (oxi-mCs)—5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)—by the TET/JBP family of dioxygenases prompted intensive studies of their functional roles in mammalian cells. However, the functional interplay of these less abundant modified nucleotides in other eukaryotic lineages remains poorly understood. We carried out a systematic study of the content and distribution of oxi-mCs in the DNA and RNA of the basidiomycetes Laccaria bicolor and Coprinopsis cinerea, which are established models to study DNA methylation and developmental and symbiotic processes. Quantitative liquid chromatography–tandem mass spectrometry revealed persistent but uneven occurrences of 5hmC, 5fC and 5caC in the DNA and RNA of the two organisms, which could be upregulated by vitamin C. 5caC in RNA (5carC) was predominantly found in non-ribosomal RNA, which potentially includes non-coding, messenger and small RNA species. Genome-wide mapping of 5hmC and 5fC using the single CG analysis techniques hmTOP-seq and foTOP-seq pointed at involvement of oxi-mCs in the regulation of gene expression and silencing of transposable elements. The implicated diverse roles of 5mC and oxi-mCs in the two fungi highlight the epigenetic importance of the latter modifications, which are often neglected in standard whole-genome bisulfite analyses.

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Single-Base Resolution Mapping Reveals Distinct 5-Formylcytidine in Saccharomyces cerevisiae mRNAs

Cited by 19 publications

References 39 publications

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

A Photo-triggered Wittig-Cyclization for Detecting and Sequencing 5-Formylcytidine in RNA

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea

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