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Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase.

Hall MP, Ho CK - Nucleic Acids Res. (2006)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA.

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.

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Effects of alanine-substitution on TbCom1 methyltransferase activity. (A) Protein purification. Aliquots (3 μg) of phosphocellulose fractions of wild-type (WT) or the indicated alanine-mutants were analyzed by SDS–PAGE. Polypeptides were visualized with Coomassie blue dye. The positions and sizes (in kDa) of co-electrophoresed marker polypeptides are indicated to the right. (B) Cap methyltransferase assays. Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet, 0.1 μM 32P-labeled cap 1 RNA-IV and 4 pmol of wild-type (WT) or mutant TbCom1. After incubation for 30 min at 27°C, samples were digested with RNase cocktail and products were resolved on a 22% denaturing polyacrylamide gel. An autoradiogram of the gel is shown. Labeled species corresponding to m7GpppGmpGp (cap 1) and m7GpppGmpGmpGp (cap 2) are indicated at the left. (C) Protein–RNA complex formation. The extent of capped RNA-binding by wild-type and mutant TbCom1 proteins were analyzed by gel mobility shift assay. Percentage of protein–RNA complex formed was plotted as a function of input protein.
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fig6: Effects of alanine-substitution on TbCom1 methyltransferase activity. (A) Protein purification. Aliquots (3 μg) of phosphocellulose fractions of wild-type (WT) or the indicated alanine-mutants were analyzed by SDS–PAGE. Polypeptides were visualized with Coomassie blue dye. The positions and sizes (in kDa) of co-electrophoresed marker polypeptides are indicated to the right. (B) Cap methyltransferase assays. Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet, 0.1 μM 32P-labeled cap 1 RNA-IV and 4 pmol of wild-type (WT) or mutant TbCom1. After incubation for 30 min at 27°C, samples were digested with RNase cocktail and products were resolved on a 22% denaturing polyacrylamide gel. An autoradiogram of the gel is shown. Labeled species corresponding to m7GpppGmpGp (cap 1) and m7GpppGmpGmpGp (cap 2) are indicated at the left. (C) Protein–RNA complex formation. The extent of capped RNA-binding by wild-type and mutant TbCom1 proteins were analyzed by gel mobility shift assay. Percentage of protein–RNA complex formed was plotted as a function of input protein.

Mentions: We selected eight residues in TbCom1 for alanine-substitution: three in the putative AdoMet-binding pocket (His75, Asp142 and Arg144) and four in the putative cap-binding pocket (Tyr18, Tyr187, Asp192 and Glu253) as indicated by asterisks in Figure 1. In addition, we mutated Val115, which is conserved among kinetoplastid Com1 proteins but is absent in VP39. The mutant TbCom1 proteins were expressed and purified in parallel with wild-type TbCom1 (Figure 6A), and their methyltransferase activities were compared to wild-type in the presence of 50 μM AdoMet and 0.1 μM cap 1-terminated RNA-IV (Figure 6B). The Y18A, H75A, D142A and R144A mutations reduced the methyltransferase activity to <2% of wild-type level. The Y187A and E253A proteins showed moderately reduced levels of activity (16 and 22% of wild-type level, respectively). In contrast, the V115A and D192A mutations retain near wild-type activities.


Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase.

Hall MP, Ho CK - Nucleic Acids Res. (2006)

Effects of alanine-substitution on TbCom1 methyltransferase activity. (A) Protein purification. Aliquots (3 μg) of phosphocellulose fractions of wild-type (WT) or the indicated alanine-mutants were analyzed by SDS–PAGE. Polypeptides were visualized with Coomassie blue dye. The positions and sizes (in kDa) of co-electrophoresed marker polypeptides are indicated to the right. (B) Cap methyltransferase assays. Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet, 0.1 μM 32P-labeled cap 1 RNA-IV and 4 pmol of wild-type (WT) or mutant TbCom1. After incubation for 30 min at 27°C, samples were digested with RNase cocktail and products were resolved on a 22% denaturing polyacrylamide gel. An autoradiogram of the gel is shown. Labeled species corresponding to m7GpppGmpGp (cap 1) and m7GpppGmpGmpGp (cap 2) are indicated at the left. (C) Protein–RNA complex formation. The extent of capped RNA-binding by wild-type and mutant TbCom1 proteins were analyzed by gel mobility shift assay. Percentage of protein–RNA complex formed was plotted as a function of input protein.
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Related In: Results  -  Collection

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fig6: Effects of alanine-substitution on TbCom1 methyltransferase activity. (A) Protein purification. Aliquots (3 μg) of phosphocellulose fractions of wild-type (WT) or the indicated alanine-mutants were analyzed by SDS–PAGE. Polypeptides were visualized with Coomassie blue dye. The positions and sizes (in kDa) of co-electrophoresed marker polypeptides are indicated to the right. (B) Cap methyltransferase assays. Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet, 0.1 μM 32P-labeled cap 1 RNA-IV and 4 pmol of wild-type (WT) or mutant TbCom1. After incubation for 30 min at 27°C, samples were digested with RNase cocktail and products were resolved on a 22% denaturing polyacrylamide gel. An autoradiogram of the gel is shown. Labeled species corresponding to m7GpppGmpGp (cap 1) and m7GpppGmpGmpGp (cap 2) are indicated at the left. (C) Protein–RNA complex formation. The extent of capped RNA-binding by wild-type and mutant TbCom1 proteins were analyzed by gel mobility shift assay. Percentage of protein–RNA complex formed was plotted as a function of input protein.
Mentions: We selected eight residues in TbCom1 for alanine-substitution: three in the putative AdoMet-binding pocket (His75, Asp142 and Arg144) and four in the putative cap-binding pocket (Tyr18, Tyr187, Asp192 and Glu253) as indicated by asterisks in Figure 1. In addition, we mutated Val115, which is conserved among kinetoplastid Com1 proteins but is absent in VP39. The mutant TbCom1 proteins were expressed and purified in parallel with wild-type TbCom1 (Figure 6A), and their methyltransferase activities were compared to wild-type in the presence of 50 μM AdoMet and 0.1 μM cap 1-terminated RNA-IV (Figure 6B). The Y18A, H75A, D142A and R144A mutations reduced the methyltransferase activity to <2% of wild-type level. The Y187A and E253A proteins showed moderately reduced levels of activity (16 and 22% of wild-type level, respectively). In contrast, the V115A and D192A mutations retain near wild-type activities.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA.

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.

Show MeSH
Related in: MedlinePlus