2'-O-methylation of the wobble residue of elongator pre-tRNAMet in Haloferax volcanii is guided by a box C/D RNA containing unique features

Abstract

The wobble residue C34 of Haloferax volcanii elongator tRNAMet is 2'-O-methylated. Neither a protein enzyme nor a guide RNA for this modification has been described. In this study, we show that this methylation is guided by a box C/D RNA targeting the intron-containing precursor of the tRNA. This guide RNA is starkly different from its homologs. This unique RNA of approximately 75 bases, named sR-tMet, is encoded in the genomes of H. volcanii and several other haloarchaea. A unique feature of sR-tMet is that the mature RNA in H. volcanii is substantially larger than its predicted size, whereas those in other haloarchaea are as predicted. While the 5'-ends of all tested haloarchaeal sR-tMets are equivalent, H. volcanii sR-tMet possesses an additional 51-base extension at its 3' end. This extension is present in the precursor but not in the mature sR-tMet of Halobacterium sp., suggesting differential 3'-end processing of sR-tMet in these two closely related organisms. Archaeal box C/D RNAs mostly contain a K-loop at the C'/D' motif. Another unique feature of sR-tMet is that its C'/D' motif lacks either a conventional K-turn or a K-loop. Instead, it contains two tandem, sheared G•A base pairs and a pyrimidine-pyrimidine pair in the non-canonical stem; the latter may form an alternative K-turn. Gel shift assays indicate that the L7Ae protein can form a stable complex with this unusual C'/D' motif, suggesting a novel RNA structure for L7Ae interaction.