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J. Biol. Chem., Vol. 279, Issue 46, 47661-47671, November 12, 2004

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Sequential 2'-O-Methylation of Archaeal Pre-tRNA^Trp Nucleotides Is Guided by the Intron-encoded but trans-Acting Box C/D Ribonucleoprotein of Pre-tRNA^*

Sanjay K. Singh, Priyatansh Gurha, Elizabeth J. Tran, E. Stuart Maxwell, and Ramesh Gupta¶

From the Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901-4413 and Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Haloferax volcanii pre-tRNA^Trp processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNA^Trp in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNA^Trp or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson-Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNA^Trp added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intron-encoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pre-tRNA^Trp intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing.

Received for publication, August 3, 2004 , and in revised form, September 3, 2004.

^* This work was supported by National Institutes of Health Grant GM55945 (to R. G.) and National Science Foundation Grant MCB-0215545 (to E. S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, IL 62901-4413. Tel.: 618-453-6466; Fax: 618-453-6440; E-mail: rgupta{at}siumed.edu.

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