Sequential 2'-O-methylation of archaeal pre-tRNATrp nucleotides is guided by the intron-encoded but trans-acting box C/D ribonucleoprotein of pre-tRNA

J Biol Chem. 2004 Nov 12;279(46):47661-71. doi: 10.1074/jbc.M408868200. Epub 2004 Sep 3.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Gene Expression Regulation, Archaeal
  • Haloferax volcanii / genetics
  • Haloferax volcanii / metabolism
  • Introns*
  • Methylation
  • Mutagenesis, Site-Directed
  • Nucleic Acid Conformation
  • RNA Precursors / chemistry
  • RNA Precursors / metabolism*
  • RNA, Archaeal / chemistry
  • RNA, Archaeal / metabolism*
  • RNA, Transfer, Trp / chemistry
  • RNA, Transfer, Trp / metabolism*
  • Ribonucleoproteins, Small Nuclear / chemistry
  • Ribonucleoproteins, Small Nuclear / genetics
  • Ribonucleoproteins, Small Nuclear / metabolism*


  • Archaeal Proteins
  • RNA Precursors
  • RNA, Archaeal
  • RNA, Transfer, Trp
  • Ribonucleoproteins, Small Nuclear