Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe

Abstract

RNA cap guanine-N2 methyltransferases such as Schizosaccharomyces pombe Tgs1 and Giardia lamblia Tgs2 catalyze methylation of the exocyclic N2 amine of 7-methylguanosine. Here we performed a mutational analysis of Giardia Tgs2, entailing an alanine scan of 17 residues within the minimal active domain. Alanine substitutions at Phe18, Thr40, Asp76, Asn103 and Asp140 reduced methyltransferase specific activity to <3% of wild-type Tgs2, thereby defining these residues as essential. Alanines at Pro142, Tyr148 and Pro185 reduced activity to 7-12% of wild-type. Structure-activity relationships at Phe18, Thr40, Asp76, Asn103, Asp140 and Tyr148, and at three other essential residues defined previously (Asp68, Glu91 and Trp143) were gleaned by testing the effects of 18 conservative substitutions. Our results engender a provisional map of the Tgs2 active site, which we discuss in light of crystal structures of related methyltransferases. A genetic analysis of S. pombe Tgs1 showed that it is nonessential. An S. pombe tgs1Delta strain grows normally, notwithstanding the absence of 2,2,7-trimethylguanosine caps on its U1, U2, U4 and U5 snRNAs. However, we find that S. pombe requires cap guanine-N7 methylation catalyzed by the enzyme Pcm1. Deletion of the pcm1(+) gene was lethal, as were missense mutations in the Pcm1 active site. Thus, whereas m(7)G caps are essential in both S. pombe and S. cerevisiae, m(2,2,7)G caps are not.

Figure 1.

Conserved Tgs2 amino acids targeted for mutagenesis. The amino acid sequence of Giardia lamblia (Gla) Tgs2 from residues 17–190 is aligned to the sequence of its paralog GlaTgs1 and to the sequences of homologous Tgs1 polypeptides encoded by Schizosaccharomyces pombe (Spo), Saccharomyces cerevisiae (Sce) and Homo sapiens (Hsa). Gaps in the alignment are indicated by dashes. Positions targeted for alanine scanning in the present study are indicated by filled circles. Residues that were subjected previously to alanine substitution (26) are indicated by verticle lines. Essential side chains are highlighted in shaded boxes.

Figure 2.

Effects of N- and C-terminal deletions on Tgs2 activity. (A) Aliquots (4 µg) of the nickel-agarose preparations of wild-type (WT) Tgs2 and deletion mutants Tgs2-(14-258), Tgs2-(31-258), and Tgs2-(1-235) were analyzed by SDS-PAGE. Polypeptides were visualized by staining the gel with Coomassie Brilliant Blue dye. A scan of the stained gel is shown. The positions and sizes (in kDa) of marker proteins are indicated on the left. (B) Methyltransferase reaction mixtures (20 µl) containing 50 µM [³H-CH₃]-AdoMet, 2.5 mM m⁷GDP and wild-type or mutant proteins as specified were incubated for 15 min at 37°C. The extent of methyl transfer per 20 µl reaction (containing 1000 pmol of input AdoMet) is plotted as a function of input enzyme.

Figure 3.

Tgs2 missense mutants. Aliquots (2 µg) of the nickel-agarose preparations of wild-type (WT) Tgs2 and the indicated Tgs2-Ala mutants (panel A) or conservative mutants (panel B) were analyzed by SDS-PAGE. A scan of the Coomassie Blue-stained gel is shown. The positions and sizes (in kDa) of marker proteins are indicated on the left or right.

Figure 4.

Effects of alanine mutations on methyltransferase activity. Methyltransferase reaction mixtures (20 µl) containing 50 µM [³H-CH₃]-AdoMet, 2.5 mM m⁷GDP and wild-type or mutant proteins as specified were incubated for 15 min at 37°C. The extent of methyl transfer per 20 µl reaction (containing 1000 pmol of AdoMet) is plotted as a function of input enzyme.

Figure 5.

Effects of conservative amino acid substitutions. Methyltransferase reaction mixtures (20 µl) containing 50 µM [³H-CH₃]-AdoMet, 2.5 mM m⁷GDP and wild-type or mutant proteins as specified were incubated for 15 min at 37°C. The extent of methyl transfer per 20 µl reaction (containing 1000 pmol of AdoMet) is plotted as a function of input enzyme.

Figure 6.

Cap guanine-N2 methylation is not essential in S. pombe. Total RNA from tgs1⁺ and tgs1Δ strains of S. pombe was subjected to immunoprecipitation with anti-TMG antibody bound to protein A sepharose beads. Aliquots of input RNA (I), immunoprecipitated RNA (P) and unbound RNA from the supernatant (S) were analyzed by northern blotting using probes to detect U1, U2, U4 and U5 snRNA and the uncapped 5.8S RNA as described in methods section. Autoradiograms of the blots are shown.

Figure 7.

Cap guanine-N7 methyltransferase is essential in S. pombe. A pcm1Δ p(ura4⁺ pcm1⁺) haploid strain of S. pombe was transformed with LEU2 plasmids bearing wild-type pcm1⁺, pcm1 mutants D145A, R181A and Y225A or wild-type S. cerevisiae ABD1. A control transformation was performed with the empty pREP81x vector. Leu⁺ transformants were tested for growth on medium containing FOA. The plates were photographed after 3 days of incubation at 30°C.