title: Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase.
abstract: Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2'-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2'-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2'-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.
authors.affiliation: Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA.
authors.forename: Megan P
authors.initials: MP
authors.lastname: Hall
authors.forename: C Kiong
authors.initials: CK
authors.lastname: Ho
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D015800:Protozoan Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D008780:Methyltransferases; C032116:cap II RNA(nucleoside-2'-)methyltransferase