Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

Abstract

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m(7)G to m(2,7)G, and (2) conversion of m(2,7)G to m(2,2,7)G. hTgs1 is unable to methylate G or m(2)G, signifying that both steps require an m(7)G cap. hTgs1 utilizes a broad range of m(7)G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl(7)G and ethyl(7)G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key pi-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.

FIGURE 1.

Methyl acceptor specificity of human Tgs1. Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 50 mM NaCl, 100 μM [³H-CH₃]AdoMet, 1 mM guanine nucleotide methyl acceptor as specified, and 1 μg of hTgs1(631–853) were incubated for 60 min at 37°C. Aliquots (6 μL) were spotted on PEI-cellulose TLC plates, which were developed either with 0.2 M (NH₄)₂SO₄ (A,D) or 0.05 M (NH₄)₂SO₄ (B,C,E). A control reaction mixture containing enzyme but no guanine nucleotide was included in panel B (lane –). The chromatograms were treated with Enhance, and ³H-labeled material was visualized by autoradiography. (Arrowheads) Positions of unlabeled markers corresponding to methyltransferase reaction products.

FIGURE 2.

Methyl acceptor specificity of Giardia Tgs2. Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 50 mM NaCl, 100 μM [³H-CH₃]AdoMet, 1 mM guanine nucleotide methyl acceptor as specified, and 0.5 μg of GlaTgs2 were incubated for 60 min at 37°C. Aliquots (6 μL) were spotted on PEI-cellulose TLC plates, which were developed either with 0.2 M (NH₄)₂SO₄ (A) or 0.05 M (NH₄)₂SO₄ (B,C). ³H-labeled material was visualized by autoradiography.

FIGURE 3.

Mimivirus Tgs catalyzes a single methyl addition at cap guanine-N2 and has relaxed cap specificity at alkaline pH. Reaction mixtures (10 μL) containing either 50 mM Tris-HCl (pH 8.0) or 50 mM Tris-acetate (pH 6.0) as indicated; 5 mM DTT, 50 mM NaCl, 100 μM [³H-CH₃]AdoMet, 1 mM methyl acceptor as specified; and 1 μg (A,B) or 0.5 μg (C) of MimiTgs were incubated for 60 min at 37°C. Aliquots (6 μL) were spotted on PEI-cellulose TLC plates, which were developed with 0.2 M (NH₄)₂SO₄ (A) or 0.05 M (NH₄)₂SO₄ (B,C). ³H-labeled material was visualized by autoradiography.

FIGURE 4.

Guanine-N2 methylation without N7 methylation. (A,B) Reaction mixtures (10 μL) containing 50 mM Tris buffer (either Tris-acetate pH 5.0–6.5 or Tris-HCl pH 7.0–9.0), 5 mM DTT, 50 mM NaCl, 100 μM [³H-CH₃]AdoMet, 1 mM GDP (A) or 1 mM GTP (B), and 1 μg of MimiTgs were incubated for 60 min at 37°C. Aliquots (6 μL) were spotted on PEI-cellulose TLC plates, which were developed either with 0.2 M (NH₄)₂SO₄ (A) or 0.4 M (NH₄)₂SO₄ (B). (C) Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 9.0), 5 mM DTT, 100 μM [³H-CH₃]AdoMet, 1 mM GTP, ATP, ITP, or UTP as specified, and 1 μg of MimiTgs were incubated for 60 min at 37°C. Aliquots (6 μL) were spotted on PEI-cellulose TLC plates, which were developed with 0.4 M (NH₄)₂SO₄. ³H-labeled material was visualized by autoradiography.