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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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TbCom1 transfers a methyl group to the 2′-OH ribose moiety of the 2nd transcribed nucleoside. (A) Capped RNA substrates. 32P-labeled 35mer cap 0 RNA substrates (RNA-I, -II, -III and -IV) were prepared by in vitro transcription as described in Materials and Methods. Asterisks indicate positions of 32P-labeled phosphates. One pmol of 32P-labeled cap 0 RNA-I, -II, -III and IV as shown in (A) was incubated in 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet for 30 min at 27°C with either 4 pmol of vaccinia VP39 (V: lanes 2, 5, 8 and 11) or TbCom1 (Tb; lanes 3, 6, 9 and 12). Control reaction in the absence of methyltransferase are indicated by (−). Reaction products were digested with either nuclease P1 (B) or an RNase cocktail (C), and products were resolved by 22% denaturing polyacrylamide gel. The autoradiographs of the gels are shown.
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fig3: TbCom1 transfers a methyl group to the 2′-OH ribose moiety of the 2nd transcribed nucleoside. (A) Capped RNA substrates. 32P-labeled 35mer cap 0 RNA substrates (RNA-I, -II, -III and -IV) were prepared by in vitro transcription as described in Materials and Methods. Asterisks indicate positions of 32P-labeled phosphates. One pmol of 32P-labeled cap 0 RNA-I, -II, -III and IV as shown in (A) was incubated in 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet for 30 min at 27°C with either 4 pmol of vaccinia VP39 (V: lanes 2, 5, 8 and 11) or TbCom1 (Tb; lanes 3, 6, 9 and 12). Control reaction in the absence of methyltransferase are indicated by (−). Reaction products were digested with either nuclease P1 (B) or an RNase cocktail (C), and products were resolved by 22% denaturing polyacrylamide gel. The autoradiographs of the gels are shown.

Mentions: To determine the site of RNA methylation by TbCom1, we synthesized three additional cap 0 RNAs containing radiolabeled phosphates at positions 1, 2 and 3 of the transcribed RNA (designated cap 0 RNA-I, -II and -III, respectively; Figure 3A). TbCom1 was incubated with these capped RNAs in the presence of AdoMet and then treated with either nuclease P1 or an RNase cocktail. The digested products were resolved on a high percentage denaturing polyacrylamide gel. In the control reaction, digestion of cap 0 RNA-I, -II, -III or -IV with nuclease P1 generated m7GpppG (Figure 3B; lanes 1, 4, 7 and 10). Addition of VP39 prior to nuclease P1 digestion shifts the m7GpppG to a slower-migrating band corresponding to the m7GpppGm cap 1 structure (Figure 3B; lanes 2, 5, 8 and 11). Incubation of TbCom1 with any of the cap 0 RNA substrates did not alter the mobility of m7GpppG (Figure 3B; lanes 3, 6, 9 and 12), consistent with the 2D TLC result that TbCom1 does not modify the nucleoside at position 1. When cap 0 RNA-II, -III or -IV was incubated with TbCom1, a novel radiolabeled species appeared which migrated between m7GpppG and pG (Figure 3B; lanes 6, 9 and 12). This radiolabeled species likely corresponds to methylated pG (pGm), as the appearance of this band was dependent on the addition of AdoMet (data not shown). Furthermore, treatment with alkaline phosphatase after nuclease P1 digestion eliminated this species (data not shown). The methyl transfer reaction was specific to cap 0- and cap 1-terminated RNA. No detectable methylation was observed using an RNA substrate lacking the cap N-7 methyl group (Figure 4A and data not shown).


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

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

TbCom1 transfers a methyl group to the 2′-OH ribose moiety of the 2nd transcribed nucleoside. (A) Capped RNA substrates. 32P-labeled 35mer cap 0 RNA substrates (RNA-I, -II, -III and -IV) were prepared by in vitro transcription as described in Materials and Methods. Asterisks indicate positions of 32P-labeled phosphates. One pmol of 32P-labeled cap 0 RNA-I, -II, -III and IV as shown in (A) was incubated in 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet for 30 min at 27°C with either 4 pmol of vaccinia VP39 (V: lanes 2, 5, 8 and 11) or TbCom1 (Tb; lanes 3, 6, 9 and 12). Control reaction in the absence of methyltransferase are indicated by (−). Reaction products were digested with either nuclease P1 (B) or an RNase cocktail (C), and products were resolved by 22% denaturing polyacrylamide gel. The autoradiographs of the gels are shown.
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Related In: Results  -  Collection

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fig3: TbCom1 transfers a methyl group to the 2′-OH ribose moiety of the 2nd transcribed nucleoside. (A) Capped RNA substrates. 32P-labeled 35mer cap 0 RNA substrates (RNA-I, -II, -III and -IV) were prepared by in vitro transcription as described in Materials and Methods. Asterisks indicate positions of 32P-labeled phosphates. One pmol of 32P-labeled cap 0 RNA-I, -II, -III and IV as shown in (A) was incubated in 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet for 30 min at 27°C with either 4 pmol of vaccinia VP39 (V: lanes 2, 5, 8 and 11) or TbCom1 (Tb; lanes 3, 6, 9 and 12). Control reaction in the absence of methyltransferase are indicated by (−). Reaction products were digested with either nuclease P1 (B) or an RNase cocktail (C), and products were resolved by 22% denaturing polyacrylamide gel. The autoradiographs of the gels are shown.
Mentions: To determine the site of RNA methylation by TbCom1, we synthesized three additional cap 0 RNAs containing radiolabeled phosphates at positions 1, 2 and 3 of the transcribed RNA (designated cap 0 RNA-I, -II and -III, respectively; Figure 3A). TbCom1 was incubated with these capped RNAs in the presence of AdoMet and then treated with either nuclease P1 or an RNase cocktail. The digested products were resolved on a high percentage denaturing polyacrylamide gel. In the control reaction, digestion of cap 0 RNA-I, -II, -III or -IV with nuclease P1 generated m7GpppG (Figure 3B; lanes 1, 4, 7 and 10). Addition of VP39 prior to nuclease P1 digestion shifts the m7GpppG to a slower-migrating band corresponding to the m7GpppGm cap 1 structure (Figure 3B; lanes 2, 5, 8 and 11). Incubation of TbCom1 with any of the cap 0 RNA substrates did not alter the mobility of m7GpppG (Figure 3B; lanes 3, 6, 9 and 12), consistent with the 2D TLC result that TbCom1 does not modify the nucleoside at position 1. When cap 0 RNA-II, -III or -IV was incubated with TbCom1, a novel radiolabeled species appeared which migrated between m7GpppG and pG (Figure 3B; lanes 6, 9 and 12). This radiolabeled species likely corresponds to methylated pG (pGm), as the appearance of this band was dependent on the addition of AdoMet (data not shown). Furthermore, treatment with alkaline phosphatase after nuclease P1 digestion eliminated this species (data not shown). The methyl transfer reaction was specific to cap 0- and cap 1-terminated RNA. No detectable methylation was observed using an RNA substrate lacking the cap N-7 methyl group (Figure 4A and data not shown).

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