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

Marchand, Virginie; Pichot, Florian; Thüring, Kathrin; Ayadi, Lilia; Freund, Isabel; Dalpke, Alexander H.; Helm, Mark; Motorin, Yuri

doi:10.3390/biom7010013

Cited by 53 publications

(61 citation statements)

References 66 publications

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“…RiboMethSeq analysis of D.melanogaster tRNAs was performed as described in (81) . Briefly, tRNAs extracted from whole flies were fragmented in 50 mM bicarbonate buffer pH 9.2 for 15 min at 95°C.…”

Section: Ribomethseqmentioning

confidence: 99%

tRNA 2’-O-methylation modulates small RNA silencing and life span in Drosophila

Angelova

Dimitrova

Silva

et al. 2019

Preprint

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2'-O-methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two novel Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1, respectively. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNA Phe , tRNA Trp and tRNA Leu , while subsequently, CG5220 methylates position C32 in the same tRNAs and targets also additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as life span reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalysing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

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Section: Ribomethseqmentioning

confidence: 99%

tRNA 2’-O-methylation modulates small RNA silencing and life span in Drosophila

Angelova

Dimitrova

Silva

et al. 2019

Preprint

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“…Recently, we published a high-throughput deep sequencingbased approach, named RiboMethSeq, for mapping of 2′-Omethylations in highly abundant RNAs, mostly in rRNA (Marchand et al, 2016;Erales et al, 2017), with possible extension to tRNA (Marchand et al, 2017a;Freund et al, 2019). This protocol is also suitable for low abundance RNAs (Krogh et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

et al. 2020

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A major trend in the epitranscriptomics field over the last 5 years has been the highthroughput analysis of RNA modifications by a combination of specific chemical treatment (s), followed by library preparation and deep sequencing. Multiple protocols have been described for several important RNA modifications, such as 5-methylcytosine (m 5 C), pseudouridine (y), 1-methyladenosine (m 1 A), and 2′-O-methylation (Nm). One commonly used method is the alkaline cleavage-based RiboMethSeq protocol, where positions of reads' 5'-ends are used to distinguish nucleotides protected by ribose methylation. This method was successfully applied to detect and quantify Nm residues in various RNA species such as rRNA, tRNA, and snRNA. Such applications require adaptation of the initially published protocol(s), both at the wet bench and in the bioinformatics analysis. In this manuscript, we describe the optimization of RiboMethSeq bioinformatics at the level of initial read treatment, alignment to the reference sequence, counting the 5′-and 3′ends, and calculation of the RiboMethSeq scores, allowing precise detection and quantification of the Nm-related signal. These improvements introduced in the original pipeline permit a more accurate detection of Nm candidates and a more precise quantification of Nm level variations. Applications of the improved RiboMethSeq treatment pipeline for different cellular RNA types are discussed.

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“…However, the method relies heavily on negative rather than positive signals. In addition, the requirement for high read depth and coverage makes such studies costly, and the method can also suffer from high background noise due to resistance to alkaline hydrolysis of highly structured regions (Marchand et al 2017). In order to address these issues, we have developed a 2 ′ -O-methyl ribose-specific, high-throughput method, which relies on positive rather than negative signals, to detect 2 ′ -O-methylation sites.…”

Section: Introductionmentioning

confidence: 99%

High-throughput and site-specific identification of 2′-O-methylation sites using ribose oxidation sequencing (RibOxi-seq)

Zhu

Pirnie

Carmichael

2017

RNA

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Ribose methylation (2 ′ ′ ′ ′ ′ -O-methylation, 2 ′ ′ ′ ′ ′ -OMe) occurs at high frequencies in rRNAs and other small RNAs and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2 ′ ′ ′ ′ ′ -Omethylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next-generation sequencing. A key feature of this method is the generation of RNA fragments with random 3 ′ ′ ′ ′ ′ -ends, followed by periodate oxidation of all molecules terminating in 2 ′ ′ ′ ′ ′ ,3 ′ ′ ′ ′ ′ -OH groups. This allows only RNAs harboring 2 ′ ′ ′ ′ ′ -OMe groups at their 3 ′ ′ ′ ′ ′ -ends to be sequenced. Although currently requiring microgram amounts of starting material, this method is robust for the analysis of rRNAs even at low sequencing depth.

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

Cited by 53 publications

References 66 publications

tRNA 2’-O-methylation modulates small RNA silencing and life span in Drosophila

tRNA 2’-O-methylation modulates small RNA silencing and life span in Drosophila

Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

High-throughput and site-specific identification of 2′-O-methylation sites using ribose oxidation sequencing (RibOxi-seq)

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