RNA 5-Methylcytosine Analysis by Bisulfite Sequencing

Schaefer, Matthias

doi:10.1016/bs.mie.2015.03.007

Cited by 53 publications

(36 citation statements)

References 114 publications

(169 reference statements)

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“…To obtain a transcriptome-wide landscape of m 5 C profiles, we performed RNA-BisSeq analysis on RNA samples purified from human HeLa cells. Based on a recently described method 29 , we used ACT random hexamers devoid of Gs to prime the reverse transcription (RT) of bisulfite-treated poly(A)-enriched RNA samples aiming to avoid copying inefficiently deaminated RNA templates. Since RNA-BisSeq cannot distinguish m 5 C from its potential oxidation product of hm 5 C, we applied UHPLC-MRM-MS/MS (ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry coupled with multiple-reaction monitoring) to verify the presence of m 5 C and hm 5 C in mammalian mRNAs.…”

Section: Resultsmentioning

confidence: 99%

5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m5C reader

et al. 2017

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5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modiﬁcation is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

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Section: Resultsmentioning

confidence: 99%

5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m5C reader

et al. 2017

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“…To determine the location of modifications with nucleotide resolution, reverse transcription (RT) and truncation approaches have also been employed. (6,(15)(16)(17) These approaches rely on manipulating either buffer conditions or the modified RNA directly, to encourage the RT enzyme to terminate prematurely upon encountering a base with either an endogenous modification or an adduct appended to a modification by a specific chemical reaction. While RT truncation-based methods provide localization information and estimates of modification quantification, they are complicated by RT-bias and cannot interrogate multiple modifications from the same single molecule.…”

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confidence: 99%

Direct detection of RNA modifications and structure using single molecule nanopore sequencing

Stephenson

Razaghi

Busan³

et al. 2020

Preprint

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Many methods exist to detect RNA modifications by short-read sequencing, relying on either antibody enrichment of transcripts bearing modified bases or mutational profiling approaches which require conversion to cDNA. Endogenous modifications are present on several major classes of RNA including tRNA, rRNA and mRNA and can modulate diverse biological processes such as genetic recoding, mRNA export and RNA folding. In addition, exogenous modifications can be introduced to RNA molecules to reveal RNA structure and dynamics. Limitations on read length and library size inherent in short-read-based methods dissociate modifications from their native context, preventing single molecule analysis and modification phasing. Here we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications over long sequence distance at the single molecule level. We demonstrate comprehensive detection of endogenous modifications in E. coli and S. cerevisiae ribosomal RNA (rRNA) using current signal deviations. Notably 2'-O-methyl (Nm) modifications generated a discernible shift in current signal and event level dwell times. We show that dwell times are mediated by the RNA motor protein which sits atop the nanopore. Further, we characterize a recently described small adduct-generating 2'-O-acylation reagent, acetylimidazole (AcIm) for exogenously labeling flexible nucleotides in RNA. Finally, we demonstrate the utility of AcIm for single molecule RNA structural probing using nanopore sequencing.2'-O-methylation (Nm) (methylation of the 2' position of the ribose sugar), is found most prominently in the 5' cap of mRNA (m 7 GpppNmNm) in eukaryotes and in ribosomal RNAs (rRNA).Recently, Nm modifications have also been detected within coding regions of mRNA (6) and have been demonstrated to have roles in tuning cognate tRNA selection during translation, thereby adjusting protein synthesis dynamics.(7) Pseudouridine ( ), often referred to as the fifth base due to its widespread inclusion in diverse classes of RNA, is generated by isomerization of uracil, thereby expanding hydrogen bonding and base pairing capabilities while also increasing base stacking propensity.(8, 9) The absence or reduction of RNA modifications has been shown to play a role in a variety of diseases including cancer (m 6 A), heart disease (m 6 A) (10), Treacher Collins syndrome (Nm) (11) and dyskeratosis congenita ( ).(12) Comprehensive detection and localization of RNA modifications within their native context would improve our understanding of RNA modification function and regulation, in addition to elucidating their role in disease. A variety of methods exist for detection of specific RNA modifications, including pulldown of RNA using antibodies (immunoprecipitation) that specifically recognize modifications (e.g. m 6 A, 5mC) (e.g. MeRIP-seq, miCLIP).(13, 14) While immunoprecipitation based approaches can be applied transcriptome-wide, they only interrogate one modification at a time, do not give nucleotide resolution and are limited by ...

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“…Still, the error rate of Illumina-based sequencing [145] might call artefacts or preclude the accurate detection of less abundant modifications or of modifications that only occur in a small subset of cells or transcripts. Chemical pre-treatment of RNAs might introduce artefacts, as has been observed for RNA bisulfite sequencing [146]. In addition, genomic SNPs might be interpreted as actual RNA modification-induced RT mismatches, and mapping reads across splice junctions or at polyadenylation sites may lead to inaccurately calling false positives, which is a major pitfall for RNA editing mapping, especially in non-model organisms.…”

Section: Where Might Rna Modifications Be?mentioning

confidence: 99%

Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’

2017

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The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the ‘epitranscriptome’. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications.

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RNA 5-Methylcytosine Analysis by Bisulfite Sequencing

Cited by 53 publications

References 114 publications

5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m5C reader

5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m5C reader

Direct detection of RNA modifications and structure using single molecule nanopore sequencing

Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’

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