The Cm56 tRNA modification in archaea is catalyzed either by a specific 2′-O-methylase, or a C/D sRNP

Abstract

We identified the first archaeal tRNA ribose 2 0 -O-methylase, aTrm56, belonging to the Cluster of Orthologous Groups (COG) 1303 that contains archaeal genes only. The corresponding protein exhibits a SPOUT S-adenosylmethionine (AdoMet)-dependent methyltransferase domain found in bacterial and yeast G18 tRNA 2 0 -O-methylases (SpoU, Trm3). We cloned the Pyrococcus abyssi PAB1040 gene belonging to this COG, expressed and purified the corresponding protein, and showed that in vitro, it specifically catalyzes the AdoMet-dependent 2 0 -O-ribose methylation of C at position 56 in tRNA transcripts. This tRNA methylation is present only in archaea, and the gene for this enzyme is present in all the archaeal genomes sequenced up to now, except in the crenarchaeon Pyrobaculum aerophilum. In this archaea, the C56 2 0 -O-methylation is provided by a C/D sRNP. Our work is the first demonstration that, within the same kingdom, two different mechanisms are used to modify the same nucleoside in tRNAs.

Characterization of two homologous 2′-O-methyltransferases showing different specificities for their tRNA substrates

“…This modification could have occurred on two positions in the tRNA: nucleotides 32 and 56 (Kuchino et al 1982). However, since the enzyme catalyzing the formation of Cm56 is known (Saci_0653 encoded protein) (Renalier et al 2005) and the replacement of C32 by U in [α 32 P]CTP-labeled C32U tRNA i Met abolishes the Cm formation by Saci_0621 (Fig. 1B), we conclude that the Saci_0621 encoded protein is responsible for 2 ′ -O-methylation at position 32 of S. acidocaldarius tRNA.…”

“…In Eukarya, mainly ribosomal RNA (rRNA) and small nuclear RNA (snRNA) are modified by guided MTases (Clouet-d'Orval et al 2005;Reichow et al 2007). In Archaea, several 2 ′ -O-MTases of rRNA and, to a limited extent, transfer RNA (tRNA) utilize the guided system (Ziesche et al 2004;Renalier et al 2005).…”

Identification and characterization of RsmE, the founding member of a new RNA base methyltransferase family

“…Greater sequence similarity can be found in specific subfamilies, such as the m During the past few years, other SAM-dependent MTases were identified and were found to have a completely different SAM binding fold, an alpha/beta knot structure (Schubert et al 2003). Two of the six families of this class, SpoU and TrmD (SPOUT members), include ribose Gm and Cm (Renalier et al 2005) or m 1 G MTases, respectively. Several additional uncharacterized E. coli genes, including yggJ, also were predicted to encode MTases with alpha/beta knot structures (Koonin and Rudd 1993;Forouhar et al 2003).…”

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

“…For example, the synthesis of Cm32 and Um32 (Cm and Um = ribose 29-O-methylation of C and U) in the anticodon loop is catalyzed by TrmJ in bacteria with a trefoil-knot structure but is catalyzed by Trm7 in eukaryotes with a Rossmann-fold structure (Purta et al 2006). The synthesis of Cm56 in the T loop (Clouet-d'Orval et al 2005;Renalier et al 2005) is catalyzed by a single Trm56 enzyme in most organisms but by an RNA-protein (RNP) complex in some archaea, where the proteins in the complex perform this reaction in conjunction with an RNA guide made up of C/D boxes. The synthesis of C35 (C, pseudouridine) in the anticodon of tRNA Tyr also involves analogous enzymes, with Pus7 acting as the single enzyme in eukaryotes and with an H/ACA-guided RNP in some archaea (Muller et al 2009).…”