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

Motorin, Yuri; Marchand, Virginie

doi:10.3390/genes9120642

Cited by 49 publications

(42 citation statements)

References 64 publications

(91 reference statements)

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“…When sequencing reads are mapped to the reference sequence, 5′-end and 3′-end coverage show a characteristic drop, resulting from protection (see Supplemental Figure S5). These profiles are subsequently merged (with –1 nt or –2 nt backshift for the 5′-end coverage) to obtain a cumulated profile used for calculation of RiboMethSeq scores ( 43 ). ( F ) RiboMethSeq analysis results showing the methylation scores at the sites G1145, U1369, G1370 for WT, GTPBP5 -KO, GTPBP5 -KO reconstituted with GTPBP5 and silencing of MRM2 (siMRM2).…”

Section: Resultsmentioning

confidence: 99%

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

Maiti

Antonická

Gingras

et al. 2020

Nucleic Acids Research

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Abstract Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.

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Section: Resultsmentioning

confidence: 99%

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

Maiti

Antonická

Gingras

et al. 2020

Nucleic Acids Research

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“…Additionally, m 5 C can be also detected by antibody-based techniques ( Figure 5B) such as m 5 C-seq and m 5 C-RIP or enzyme mediated approaches ( Figure 5D) such as Aza-IP and methylation i-CLIP (Edelheit et al 2013;Hussain et al 2013;Khoddami and Cairns 2013). In conclusion, the detection of 2′-O-Me is currently performed by methods based on pretreatment with chemical reagents ( Figure 5C) such as RibOxi-seq and Nm-seq (Zhu et al 2017;Dai et al 2017;Motorin and Marchand 2018). Technologies (ONT) platform (ONT-signature).…”

Section: Detection Methodsmentioning

confidence: 98%

A mark of disease: how mRNA modifications shape genetic and acquired pathologies

Destefanis

Avşar

Groza

et al. 2020

RNA

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RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). In mRNAs the deposition, removal, and recognition of chemical marks by writers, erasers and readers influence their structure, localization, stability, and translation. In turn, this modulates key molecular and cellular processes such as RNA metabolism, cell cycle, apoptosis, and others. Unsurprisingly, given their relevance for cellular and organismal functions, alterations of epitranscriptomic marks have been observed in a broad range of human diseases, including cancer, neurological and metabolic disorders. Here, we will review the major types of mRNA modifications and editing processes in conjunction with the enzymes involved in their metabolism and describe their impact on human diseases. We present the current knowledge in an updated catalog. We will also discuss the emerging evidence on the crosstalk of epitranscriptomic marks and what this interplay could imply for the dynamics of mRNA modifications. Understanding how this complex regulatory layer can affect the course of human pathologies will ultimately lead to its exploitation toward novel epitranscriptomic therapeutic strategies.

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“…Deep sequencing-based identification of the B. subtilis tRNA modifications was performed using RiboMethSeq protocol, allowing us to map 2′-O-methylations by their protection to cleavage [ 41 , 42 ]. Reverse Transcriptase (RT) misincorporation signatures at RT-arresting nucleotides [ 43 , 44 ] were extracted from RiboMethSeq data and also from the RT-primer extension, using TTO-based ScriptSeq v2 (Illumina, San Diego, CA, USA) library preparation kit. RiboMethSeq analysis of tRNAs was performed using alkaline fragmentation of total B. subtilis RNA, followed by library preparation and sequencing [ 42 ].…”

Section: Methodsmentioning

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

Cited by 49 publications

References 64 publications

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

A mark of disease: how mRNA modifications shape genetic and acquired pathologies

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

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