Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase
MP Hall, CK Ho - Nucleic acids research, 2006 - academic.oup.com
MP Hall, CK Ho
Nucleic acids research, 2006•academic.oup.comKinetoplastid 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 …
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 …
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.
Oxford University Press
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