Detection of ribonucleoside modifications by liquid chromatography coupled with mass spectrometry

Jora, Manasses; Lobue, Peter A.; Ross, Robert L; Williams, Brittney; Addepalli, Balasubrahmanyam

doi:10.1016/j.bbagrm.2018.10.012

Cited by 52 publications

(40 citation statements)

References 122 publications

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“…However, the exact role of each ribose 2′- O -methylation remained elusive, because its labile chemical nature limits sensitivity and precludes mapping in different RNAs. The current advances in high-throughput detection of 2′- O -methylation include chromatography and mass-spectroscopy ( 60 , 61 ), RTL-P-2′- O -Me-Seq ( 62 ), and RibOxi-Seq ( 63 ), etc. All these traditional methods are not particularly specific for 2′- O -methyl modified residues and are extremely laborious and time-consuming.…”

Section: Discussionmentioning

confidence: 99%

Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

Abula

Quan

et al. 2021

Nucleic Acids Research

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RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.

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Section: Discussionmentioning

confidence: 99%

Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

Abula

Quan

et al. 2021

Nucleic Acids Research

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“…Several observed modifications must be artifacts of the sample preparation. m 1 I has only been observed in archaeal tRNA [ 62 ] but could arise from the chemical or enzymatic deamination of m 1 A during the enzymatic digestion to nucleosides [ 63 ]. Similarly, oxidation inside the cell or during analysis is most certainly the source of 8-oxoG formation [ 64 ].…”

Section: Resultsmentioning

confidence: 99%

Survey and Validation of tRNA Modifications and Their Corresponding Genes in Bacillus subtilis sp Subtilis Strain 168

et al. 2020

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Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum. Bacillus subtilis sp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted from B. subtilis, combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared to E. coli. In general, B. subtilis tRNAs are less modified than those in E. coli, even if some modifications, such as m1A22 or ms2t6A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer from E. coli to B. subtilis based on homology only without further experimental validation. This difficulty was shown with the identification of the B. subtilis enzyme that introduces ψ at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes in B. subtilis, identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria.

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“…Similar to all other modified nucleotides in RNAs, 2′- O -Me can be also detected by various types of chromatography [15,16] as well as by techniques of mass-spectrometry [17,18,19,20], which are not particularly specific for those modified residues, but still allow their detection and, in some instances, quantification (reviewed in [21,22,23]).…”

Section: Classical Methods For 2′-o-methylation Detectionmentioning

confidence: 99%

Detection and Analysis of RNA Ribose 2′-O-Methylations: Challenges and Solutions

Motorin

Marchand

2018

Genes

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Ribose 2′-O-methylation is certainly one of the most common RNA modifications found in almost any type of cellular RNA. It decorates transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs) (and most probably small nucleolar RNAs, snoRNAs), as well as regulatory RNAs like microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), and finally, eukaryotic messenger RNAs (mRNAs). Due to this exceptional widespread of RNA 2′-O-methylation, considerable efforts were made in order to precisely map these numerous modifications. Extensive studies of RNA 2′-O-methylation were also stimulated by the discovery of C/D-box snoRNA-guided machinery, which insures site-specific modification of hundreds 2′-O-methylated residues in archaeal and eukaryotic rRNAs and some other RNAs. In this brief review we discussed both traditional approaches of RNA biochemistry and also modern deep sequencing-based methods, used for detection/mapping and quantification of RNA 2′-O-methylations.

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Detection of ribonucleoside modifications by liquid chromatography coupled with mass spectrometry

Cited by 52 publications

References 122 publications

Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

Survey and Validation of tRNA Modifications and Their Corresponding Genes in Bacillus subtilis sp Subtilis Strain 168

Detection and Analysis of RNA Ribose 2′-O-Methylations: Challenges and Solutions

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