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Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme

Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
RNA (Impact Factor: 4.62). 09/1999; 5(8):1105-18. DOI: 10.1017/S1355838299982201
Source: PubMed

ABSTRACT Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w.

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    • "the intersection between the variable loop ( VL ) and the TΨC arm ( VL junction ) ( 48 / 49 / 50 ) ( Fig 1C and D , and Supplemen - tary Fig S2A ) ( Motorin et al , 2010 ; Squires et al , 2012 ; Tuorto et al , 2012 ; Khoddami & Cairns , 2013 ) . As previously reported , methyla - tion at C34 and C48 / 49 / 50 was solely dependent on NSun2 ( Fig 1D ) ( Motorin & Grosjean , 1999 ; Brzezicha et al , 2006 ; Blanco et al , 2011 ; Martinez et al , 2012 ; Squires et al , 2012 ; Tuorto et al , 2012 ; Khoddami & Cairns , 2013 ) . Methylation of C38 is mediated by Dnmt2 ( Goll et al , 2006 ) and remained unchanged when NSun2 was deleted ( Fig 1D ; Asp GTC ) . "
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    • "It is likely that Trm4 is responsible for the additional m 5 C modifications observed in tRNA His under these conditions, for two reasons. First, Trm4 is the only known S. cerevisiae m 5 C methyltransferase and can catalyze m 5 C formation on substrate tRNAs at C 34 , C 40 , C 48 , and C 49 (Motorin and Grosjean 1999). Second, trm4-Δ mutants depleted of Thg1 lack all m 5 C, including the additional m 5 C found in tRNA His at C 48 and C 50 (Gu et al. 2005). "
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    • "The protein was localized to the nucleolus and found to be nonessential (Wu et al., 1998). In 1999, the NCL1 gene was determined to be a Trm candidate in a sequence homology search against the E. coli rRNA methyltransferase SUN (Motorin and Grosjean, 1999; Tscherne et al., 1999). The Trm activity was confirmed both in vitro using scTrm4 recombinant protein and in vivo by gene replacement. "
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