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: 4.94). 09/1999; 5(8):1105-18. DOI: 10.1017/S1355838299982201
Source: PubMed


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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    • "This result is consistent with the homology between IKAP and the Elp1 subunit of the S. cerevisiae elongator complex [Hawkes et al., 2002], which is required for cm 5 U formation [Huang et al., 2005]. Second, tRNA from cells derived from patients with a Dubowitz-like syndrome (characterized by phenotypes including microcephaly and mental and speech delays) linked to an NSUN2 splice-site mutation lacked m 5 C at residues 34, 48, 49, and 50 [Martinez et al., 2012; Blanco et al., 2014], consistent with the activity of the yeast and mammalian homologs [Motorin and Grosjean, 1999; Brzezicha et al., 2006; Blanco et al., 2011; Tuorto et al., 2012]. NSUN2 mutations are also linked to other neurological disorders including a Noonan-like syndrome similar to the NSUN2-linked Dubowitz-like syndrome [Fahiminiya et al., 2014] and autosomal-recessive ID (ARID) in four independent families [Abbasi-Moheb et al., 2012; Khan et al., 2012]. "
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    ABSTRACT: tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32 ) and Gm34 on tRNA(Phe) , catalyzed by Trm7-Trm732 and Trm7-Trm734 respectively, which in turn results in loss of wybutosine at G37 . Mutations in human FTSJ1, the likely TRM7 homolog, cause non-syndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown. Here we report that tRNA(Phe) from two genetically independent cell lines of NSXLID patients with loss of function FTSJ1 mutations nearly completely lacks Cm32 and Gm34 , and has reduced peroxywybutosine (o2yW37 ). Additionally, tRNA(Phe) from an NSXLID patient with a novel FTSJ1-p.A26P missense allele specifically lacks Gm34 , but has normal levels of Cm32 and o2yW37 . tRNA(Phe) from the corresponding Saccharomyces cerevisiae trm7-A26P mutant also specifically lacks Gm34 , and the reduced Gm34 is not due to weaker Trm734 binding. These results directly link defective 2'-O-methylation of the tRNA anticodon loop to FTSJ1 mutations, suggest that the modification defects cause NSXLID, and may implicate Gm34 of tRNA(Phe) as the critical modification. These results also underscore the widespread conservation of the circuitry for Trm7-dependent anticodon loop modification of eukaryotic tRNA(Phe) . This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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    • "In archaeal tRNA-TrpCCA precursor, a box C/D small RNA is nested in the intron, and this intronic part of pre-tRNA-TrpCCA or the excised intron is used to select nucleotides C34 and U39 for 2′-O-methylation in trans (Figure 5C) (Omer et al., 2000; Clouet d'Orval et al., 2001; Singh et al., 2004). Although these modifications in eukaryotes and archaea seem to be the driving force to maintain the introns in tRNA genes, the fact that Pus1 and Trm4 are dispensable for viability of S. cerevisiae is against this assumption (Simos et al., 1996; Motorin and Grosjean, 1999). It was also demonstrated that the intron of tRNA-TrpCCA can be removed from the genome of Haloferax volcanii, suggesting that modification assisted by the intronic sequence itself does not cause strong selective pressure for intron maintenance (Joardar et al., 2008). "
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    ABSTRACT: Introns are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3' to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages.
    Full-text · Article · Jul 2014 · Frontiers in Genetics
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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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    ABSTRACT: Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5′ tRNA-derived small RNA fragments. Accumulation of 5′ tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site-specific NSun2-mediated methylation and that the presence of 5′ tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2-deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2-mediated tRNA methylation contributes to human diseases via stress-induced RNA cleavage.
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