The human tRNA m<sup>5</sup>C methyltransferase Misu is multisite-specific

Auxilien, Sylvie; Guérineau, Vincent; Szweykowska-Kulińska, Zofia; Golinelli‐Pimpaneau, Béatrice

doi:10.4161/rna.22180

Cited by 46 publications

(59 citation statements)

References 40 publications

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“…Interestingly, most of the NSUN proteins carry the putative 5-cytosine methyltransferase domain containing two catalytic cysteines (10). Surprisingly, apart from NSUN2, the human homolog of yeast Trm4, the enzymatic activity of other NSUN members has never been examined (48). …”

Section: Discussionmentioning

confidence: 99%

Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Sharma

Yang

Watzinger

et al. 2013

Nucleic Acids Research

163

211

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Yeast 25S rRNA was reported to contain a single cytosine methylation (m5C). In the present study using a combination of RP-HPLC, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m5C residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m5C residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m5C residues. The loss of m5C2278 results in anisomycin hypersensitivity, whereas the loss of m5C2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture.

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Section: Discussionmentioning

confidence: 99%

Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Sharma

Yang

Watzinger

et al. 2013

Nucleic Acids Research

163

211

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“…Cytosines are color-coded based on the number of tRNA types that we found to be NSUN2 target sites: yellow, one tRNA type; blue, 2-5 tRNA types; purple, 6-9 tRNA types; and red >10 tRNA types. For each position, the individual tRNAs are designated by their single letter amino acid abbreviation, grouped in square brackets; purple letters refer to previously known NSUN2 target sites in the designated tRNA types (in human) 2, 11, 26 . For clarity of presentation, only selected positions are depicted (See Supplementary File 5 for all tRNAs, their isoacceptors/isodecoders, and target positions).…”

Section: Figurementioning

confidence: 99%

“…Transversion was clear and significant (Fig. 3b and Supplementary File 5) at the known human NSUN2 tRNA targets (tRNA Leu (C34), tRNA Asp (C48, 49) and tRNA Gly (C48, 49, 50)) 2, 11, 26 , as well as at C48, C49 and C50 within most tRNAs (tRNA Asn and tRNA SeC excepted), greatly expanding the known repertoire. Importantly, although the target tRNAs of DNMT2 were robustly enriched within anti-V5-NSUN2 immunoprecipitates, we did not observe C>G transversion at the DNMT2 target site (C38) within the anti-V5-NSUN2 immunoprecipitates, highlighting the specificity of both the enzyme and the Aza-IP technique (Fig.…”

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confidence: 99%

“…Previous studies identified three direct tRNA targets of human NSUN2 (tRNA Leu (C34), tRNA Asp (C48, 49) and tRNA Gly (C48, 49, 50)) 2, 11, 26 , four direct tRNA targets of mouse Nsun2 (tRNA Leu (C34); tRNA Asp (C48, 49), tRNA Gly (C40, 48, 49, 50) and tRNA Val (C48, 49)) 10 , and seven additional target cytosines in the tRNA targets for yeast Trm4/Nsun2 27, 28 (Fig. 3b and Supplementary Fig.…”

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confidence: 99%

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Identification of direct targets and modified bases of RNA cytosine methyltransferases

2013

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The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a mechanism-based technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and non-coding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C>G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of cytosine RNA methyltransferases, Aza-IP may be generally applicable for target identification.

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“…In protozoan parasites, most tRNAs display at least one m 5 C at the junction between the VL and the TSL. The m 5 C modification leads to stabilization of the tRNA core in eukaryotes and archaeal tRNAs, but not in eubacteria [58]. In Trypanosoma brucei, methylation of tRNA Asp GUC , tRNA Gly GCC , tRNA Val AAC and tRNA Tyr GUA was observed in residues at the TSL/VL junction including C 48 and C 49 .…”

Section: Position 48 and 49 (M 5 C 48 And M 5 C 49 )mentioning

confidence: 99%

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function

et al. 2017

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RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA’s cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.

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The human tRNA m⁵C methyltransferase Misu is multisite-specific

Cited by 46 publications

References 40 publications

Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Identification of direct targets and modified bases of RNA cytosine methyltransferases

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function

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The human tRNA m5C methyltransferase Misu is multisite-specific

Cited by 46 publications

References 40 publications

Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively

Identification of direct targets and modified bases of RNA cytosine methyltransferases

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function

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The human tRNA m⁵C methyltransferase Misu is multisite-specific