High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA

Tserovski, Lyudmil; Marchand, Virginie; Hauenschild, Ralf; Blanloeil-Oillo, Florence; Helm, Mark; Motorin, Yuri

doi:10.1016/j.ymeth.2016.02.012

Cited by 51 publications

(40 citation statements)

References 32 publications

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“…The levels of known tRNA methylations including m 1 A, m 7 G, m 5 C, and m 3 C, were measured with ALKBH1 treatment in the presence or absence of EDTA (EDTA chelates iron and inhibits the demethylation activity) using liquid chromatography-tandem mass spectrometry (LC-MS/MS, Figure S1D) (Jia et al, 2011; Zheng et al, 2013). Incubation of total tRNA with ALKBH1 led to a dramatic decrease of the m 1 A level, but not levels of m 7 G, m 5 C, or m 3 C, in comparison to the control samples (Figure 1D and Figure S2A), suggesting that m 1 A58 in tRNAs is a major substrate of ALKBH1 (Figure 1E); m 1 A58 has been shown to be the predominant m 1 A modifications in tRNAs as revealed by recent m 1 A sequencing experiments (Cozen et al, 2015; Tserovski et al, 2016). …”

Section: Resultsmentioning

confidence: 79%

ALKBH1-Mediated tRNA Demethylation Regulates Translation

Liu

Clark

Luo

et al. 2016

Cell

423

304

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Summary Transfer RNA (tRNA) is a central component of protein synthesis and cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and their decreased usage in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new regulatory mechanism of post-transcriptional gene expression.

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Section: Resultsmentioning

confidence: 79%

ALKBH1-Mediated tRNA Demethylation Regulates Translation

Liu

Clark

Luo

et al. 2016

Cell

423

304

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“…m 1 A has been identified in toto in mRNA by liquid chromatography-tandem mass spectrometry, whereas antibody-based pulldowns coupled with next-generation sequencing have been used to map m 1 A-containing sequences genome-wide (24,59). m 1 A modifies the WC face; thus, it can also be identified by its induction of a combined stop and mismatch profile in reverse transcription (112). Biological implications of structural signatures associated with ψ, m 6 A, and m 1 A are described below.…”

Section: Methods To Identify Natural Rna Modifications Genome-widementioning

confidence: 99%

Genome-Wide Analysis of RNA Secondary Structure

Bevilacqua¹,

Ritchey²,

et al. 2016

Annu. Rev. Genet.

211

157

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Single-stranded RNA molecules fold into extraordinarily complicated secondary and tertiary structures as a result of intramolecular base pairing. In vivo, these RNA structures are not static. Instead, they are remodeled in response to changes in the prevailing physicochemical environment of the cell and as a result of intermolecular base pairing and interactions with RNAbinding proteins. Remarkable technical advances now allow us to probe RNA secondary structure at single-nucleotide resolution and genome-wide, both in vitro and in vivo. These data sets provide new glimpses into the RNA universe. Analyses of RNA structuromes in HIV, yeast, Arabidopsis, and mammalian cells and tissues have revealed regulatory effects of RNA structure on messenger RNA (mRNA) polyadenylation, splicing, translation, and turnover. Application of new methods for genome-wide identification of mRNA modifications, particularly methylation and pseudouridylation, has shown that the RNA "epitranscriptome" both influences and is influenced by RNA structure. In this review, we describe newly developed genome-wide RNA structure-probing methods and synthesize the information emerging from their application.

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“…Recently, high-throughput techniques have been developed for the analysis of a small subset of RNA modifications, namely m 6 A, m 1 A, pseudouridine and m 5 C [58,59,60,61,62]. These techniques allow transcriptome-wide screening, but the quantification of modification is uncertain and, when possible, requires complex calibration procedures.…”

Section: Introductionmentioning

confidence: 99%

Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation

et al. 2017

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Analysis of RNA modifications by traditional physico-chemical approaches is labor intensive, requires substantial amounts of input material and only allows site-by-site measurements. The recent development of qualitative and quantitative approaches based on next-generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing-based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′-O-methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally-important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′-O-methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species.

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High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA

Cited by 51 publications

References 32 publications

ALKBH1-Mediated tRNA Demethylation Regulates Translation

ALKBH1-Mediated tRNA Demethylation Regulates Translation

Genome-Wide Analysis of RNA Secondary Structure

Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation

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