tRNAs are transcribed as precursor molecules by RNA polymerase III, then the primary transcripts undergo posttranscriptional maturation before becoming functional. tRNA maturation includes the removal of leader and trailer sequences, the addition of a CCA at the 3Ј end, sometimes the splicing of an intron, and the modification of numerous nucleotides (34,42,57). In yeast tRNA, modified nucleotides are detected at 35 different positions out of 76. They are formed posttranscriptionally and require various types of enzymatic activities such as 2Ј-O-ribose methyltransferases (MTases), base MTases, pseudouridylases, deaminases, thiolases, reductases, and oxidases. Base and ribose methylations are by far the most frequent modifications in tRNA (26,57). In yeast, cytoplasmic tRNAs sequenced so far contain 15 methylated bases and five 2Ј-O-methylriboses, while mitochondrially encoded tRNA possess only 4 methylated bases, none of which are unique to mitochondria (57). It is likely that the formation of all these methylated nucleotides is catalyzed by unisite-or multisitespecific MTases. The first identified yeast tRNA MTase (Trm) Trm1p, is required for the formation of N 2 ,N 2 -dimethylguanosine at position 26 (m 2 2 G26) in cytoplasmic and mitochondrial tRNA (19). Later, several tRNA MTases were identified, by either genetic screening, bioinformatic analysis, or biochemical genomics (34). Out of the 10 tRNA MTases that have been characterized to date in yeast, only Trm6p and Trm61p, which form a heterodimer required for the formation of m 1 A58, are essential for cell growth (2). Deletion of either TRM5 or TRM7 leads to a severe growth defect (8, 51), while the genes encoding other Trm proteins can be deleted without apparent cell growth defects under laboratory conditions.With the exception of only one tetrahydrofolate-dependent enzyme (17), all known MTases acting on nucleic acids use S-adenosyl-L-methionine (AdoMet) as a cofactor (10,14). Although AdoMet-dependent MTases can be classified into at least six unrelated superfamilies based on structural and evolutionary considerations (54), all DNA MTases and most RNA MTases belong to the largest class I of Rossmann fold-like MTases (RFMs) (11). A characteristic feature of these enzymes is the presence of nine common motifs that map onto the catalytic face of the common fold, with motifs I to III involved in AdoMet binding, and motif IV and often motifs VI, VIII, and X involved in binding to the target nucleotide and in methyl transfer reactions (20).Using a combination of protein fold recognition and modeling-based identification of potential catalytic and RNA-binding residues, we previously identified 20 putative RNA MTases (termed MTase candidates or Mtcs) (14) and then tested their involvement in tRNA methylation. Here, we report the characterization of the activity responsible for the formation of m 2 G10 in yeast tRNA. The enzyme is composed of at least two subunits: Trm11p is the catalytic subunit that contains the RFM domain, and Trm112p shows similarity to an u...

rRNA molecules undergo extensive posttranscriptional modification, predominantly 2'-O-ribose methylation and pseudouridine formation, both of which are guided by the numerous small nucleolar RNAs in eukaryotes. Here, we describe an exception to this rule. The essential yeast nucleolar protein Spb1p is a site-specific rRNA methyltransferase modifying the universally conserved G2922 that is located within the A loop of the catalytic center of the ribosome. The equivalent position in bacteria is the docking site for aminoacyl-tRNA, and it is critical for translation. In sharp contrast to other 2'-O-methylriboses that are formed on the primary transcript, Gm2922 appears at a late processing stage, during the maturation of the 27S pre-rRNA. Thus, eukaryotes have maintained a site-specific enzyme to catalyze the methylation of a nucleotide that plays a crucial role in ribosome biogenesis and translation.

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 ( . An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.

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