Publications

• Pleiotropic effects of intron removal on base modification pattern of yeast tRNAPhe: an in vitro study.

  Jiang H.Q., Motorin Y., Jin Y.X., Grosjean H.

  Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of 11 out of the 14 naturally occurring modified nucleotides in yeast tRNAPhe(anticodon GAA). They are m2G10, D17, m22G26, Cm32, Gm34,psi39, m5C40, m7G46, m5C49, T54 andpsi55. Only D16, Y37 and m1A58 were not fo ... >> More

  Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of 11 out of the 14 naturally occurring modified nucleotides in yeast tRNAPhe(anticodon GAA). They are m2G10, D17, m22G26, Cm32, Gm34,psi39, m5C40, m7G46, m5C49, T54 andpsi55. Only D16, Y37 and m1A58 were not formed under in vitro conditions. However, m1G37was quantitatively produced instead of Y37. The naturally occurring intron was absolutely required for m5C40formation while it hindered completely the enzymatic formation of Cm32, Gm34and m1G37. Enzymatic formation of m22G26,psi39, m7G46, m5C49, T54 andpsi55were not or only slightly affected by the presence of the intron. These results allow us to classify the different tRNA modification enzymes into three groups: intron insensitive, intron dependent, and those requiring the absence of the intron. The fact that truncated tRNAPheconsisting of the anticodon stem and loop prolonged with the 19 nucleotide long intron is a substrate for tRNA: cytosine-40 methylase demonstrates that the enzyme is not only strictly intron dependent, but also does not require fully structured tRNA. << Less

  Nucleic Acids Res 25:2694-2701(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

  Strobel M.C., Abelson J.

  The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-me ... >> More

  The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase. << Less

  Mol Cell Biol 6:2663-2673(1986) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

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

  Motorin Y., Grosjean H.

  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) ... >> More

  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. << Less

  RNA 5:1105-1118(1999) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase.

  Walbott H., Husson C., Auxilien S., Golinelli-Pimpaneau B.

  Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulf ... >> More

  Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m(5)C967 methyltransferase, but whether this conclusion applies to other RNA m(5)C methyltransferases remains to be verified. Yeast tRNA m(5)C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex. << Less

  RNA 13:967-973(2007) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.