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.
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.
Singh
Sanjay K
SK
Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901-4413, USA.
Gurha
Priyatansh
P
Tran
Elizabeth J
EJ
Maxwell
E Stuart
ES
Gupta
Ramesh
R
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D019848:Gene Expression Regulation, Archaeal; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism; D007438:Introns; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D012364:RNA, Transfer, Trp / Q000737:chemistry / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D012322:RNA Precursors; D019642:RNA, Archaeal; D012364:RNA, Transfer, Trp; D017411:Ribonucleoproteins, Small Nuclear