Genetic and biochemical analysis of yeast and human cap trimethylguanosine synthase: functional overlap of 2,2,7-trimethylguanosine caps, small nuclear ribonucleoprotein components, pre-mRNA splicing factors, and RNA decay pathways

Abstract

Trimethylguanosine synthase (Tgs1) is the enzyme that converts standard m(7)G caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal small nuclear RNAs. Fungi and mammalian somatic cells are able to grow in the absence of Tgs1 and TMG caps, suggesting that an essential function of the TMG cap might be obscured by functional redundancy. A systematic screen in budding yeast identified nonessential genes that, when deleted, caused synthetic growth defects with tgs1Delta. The Tgs1 interaction network embraced proteins implicated in small nuclear ribonucleoprotein function and spliceosome assembly, including Mud2, Nam8, Brr1, Lea1, Ist3, Isy1, Cwc21, and Bud13. Complementation of the synthetic lethality of mud2Delta tgs1Delta and nam8Delta tgs1Delta strains by wild-type TGS1, but not by catalytically defective mutants, indicated that the TMG cap is essential for mitotic growth when redundant splicing factors are missing. Our genetic analysis also highlighted synthetic interactions of Tgs1 with proteins implicated in RNA end processing and decay (Pat1, Lsm1, and Trf4) and regulation of polymerase II transcription (Rpn4, Spt3, Srb2, Soh1, Swr1, and Htz1). We find that the C-terminal domain of human Tgs1 can function in lieu of the yeast protein in vivo. We present a biochemical characterization of the human Tgs1 guanine-N2 methyltransferase reaction and identify individual amino acids required for methyltransferase activity in vitro and in vivo.

FIGURE 1.

Mutational synergy of tgs1 with genes involved in splicing and RNA decay. Aliquots (2 μl) of serial 10-fold dilutions of haploid yeast strains of the specified genotypes were spotted on YPD agar medium. The plates were photographed after incubation for 2 days (37, 34, and 30 °C), 3 days (25 and 23° C), or 4 days (20 °C) as specified.

FIGURE 2.

Mutational synergy of tgs1, including genes involved in transcription. Aliquots (2 μl) of serial 10-fold dilutions of haploid yeast strains of the specified genotypes were spotted on YPD agar medium. The plates were photographed after incubation for 2 days (37, 34, and 30 °C), 3 days (25 and 23° C), or 4 days (20 °C) as specified.

FIGURE 3.

Mutational synergy of tgs1 with genes involved in RNA stability and transcription. Aliquots (2 μl) of serial 10-fold dilutions of haploid yeast strains of the specified genotypes were spotted on YPD agar medium. The plates were photographed after incubation for 2 days (37, 34, and 30 °C), 3 days (25 °C), or 4 days (20 °C) as specified.

FIGURE 4.

Complementation of tgs1Δ mud2Δ by yeast and human TGS1. A, yeast tgs1Δmud2Δ p360-TGS1 (URA3 CEN TGS1) cells were transformed with CEN LEU2 plasmids harboring wild-type TGS1 (positive control) or the mutant alleles D103A and D126A, and with 2μ LEU2 plasmids carrying hTGS1-(576–853), hTGS1-(607–853), or hTGS1-(631–853). Leu⁺ transformants were selected at 30 °C and then streaked to agar medium containing FOA. Cells transformed with the empty 2μ LEU2 vector served as a negative control. The plate was photographed after 4 days at 30 °C. B, serial 10-fold dilutions of tgs1Δmud2Δ cells harboring LEU2 plasmids with yeast TGS1 (positive control), wild-type hTGS1-(631–853), or the hTGS1-(631–853) T673A, S763A, and N808A mutant alleles were spotted on YPD agar medium. The plate was photographed after 3 days at 30 °C. C, amino acid sequence of Homo sapiens (Hsa) Tgs1 from residues 654 to 811 is aligned to the sequences of homologous polypeptides encoded by S. cerevisiae (Sce), S. pombe (Spo), and G. lamblia (Gla). Gaps in the alignment are indicated by dashes. Positions of identity/similarity in all four proteins are indicated by dots. Positions in hTgs1 that were targeted for alanine scanning are indicated by |.

FIGURE 5.

Methyltransferase activity of recombinant hTgs1-(576–853). A, purification. Aliquots (10 μl) of the soluble bacterial lysate (L), the nickel-agarose flow-through (FT), and the 10, 20, 50, 100, 250, and 500 mm imidazole eluate fractions were analyzed by SDS-PAGE. The polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (kDa) of marker polypeptides are indicated on the left. B, velocity sedimentation was performed as described under “Experimental Procedures.” Aliquots (18 μl) of the odd-numbered glycerol gradient fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the upper panel. The identities of the polypeptides are indicated. The methyltransferase activity profile is shown in the lower panel. Reaction mixtures (10 μl) containing 2.5 mm m⁷GpppA, 6.7 μm [³H-CH₃]AdoMet, and 1.5 μl of the indicated glycerol gradient fractions were incubated for 15 min at 37 °C. cyt c, cytochrome c.

FIGURE 6.

Methyl acceptor specificity of hTgs1. Methyltransferase reaction mixtures (20 μl) containing 50 μm [³H-CH₃]AdoMet, 4 μg of hTgs1-(576–853), and 2.5 mm cap dinucleotide (A) or nucleoside diphosphate (B) as specified were incubated for 15 min at 37 °C. Nucleotide was omitted from the control reaction in lane -. Aliquots (4 μl) were spotted onto PEI-cellulose TLC plates. The anionic nucleotides were resolved from AdoMet by ascending TLC in 50 mm ammonium sulfate. The chromatograms were treated with Enhance (PerkinElmer Life Sciences), and ³H-labeled material was visualized by autoradiography. The methyltransferase reaction products m^2,7GpppG and m^2,7GpppA (A) or m^2,7GDP (B) are denoted by ◂.

FIGURE 7.

Characterization of the hTgs1 methyltransferase reaction. A, kinetics. Reaction mixtures (100 μl) containing 1 mm m⁷GDP, 50 μm [³H-CH₃]AdoMet, and either 2.5 μg (□) or 10 μg (▪) of hTgs1-(576–853) were incubated at 37 °C. Aliquots (4 μl) were withdrawn at the times indicated, and product formation was analyzed by TLC. B, m⁷GDP dependence. Reaction mixtures (20 μl) containing 50 μm [³H-CH₃]AdoMet, 200 ng of hTgs1-(576–853), and m⁷GDP as specified were incubated for 15 min at 37 °C. C, AdoMet dependence. Reaction mixtures (20 μl) containing 1 mm m⁷GDP, 100 ng hTgs1-(576–853), and [³H-CH₃]AdoMet as specified were incubated for 15 min at 37 °C. D, inhibition by AdoHcy. Reaction mixtures (20 μl) containing 1 mm m⁷GDP, 50 μm [³H-CH₃]AdoMet, 300 ng hTgs1-(576–853), and AdoHcy as specified were incubated for 15 min at 37 °C.

FIGURE 8.

Trimethylguanosine synthesis by Tgs1. A reaction mixture containing 1 mm m⁷GDP, 50 μm [³H-CH₃]AdoMet, and 2.2 μm hTgs1-(576–853) was incubated for 15 min at 37 °C (pulse) and then supplemented with 1 mm unlabeled AdoMet and 6.8 μm hTgs1-(576–853) (chase). Aliquots (4 μl) were withdrawn at the times indicated (relative to initiation of the pulse reaction) and then spotted onto PEI-cellulose TLC plates. The products were analyzed by ascending TLC in 100 mm ammonium sulfate. The chromatogram was treated with Enhance (PerkinElmer Life Sciences), and ³H-labeled material was visualized by autoradiography. The portion of the chromatogram containing the guanosine nucleotides is shown.

FIGURE 9.

Effects of N- and C-terminal deletions on hTgs1 methyltransferase activity. A, aliquots (4 μg) of the nickel-agarose preparations of deletion mutants Tgs1-(576–853), Tgs1-(607–853), Tgs1-(631–853), Tgs1-(662–853), and Tgs1-(631–846) were analyzed by SDS-PAGE. Polypeptides were visualized by staining the gel with Coomassie Brilliant Blue dye. The positions and sizes (kDa) of marker proteins are indicated on the left. B, methyltransferase reaction mixtures (20 μl) containing 50 μm [³H-CH₃]AdoMet, 1 mm m⁷GDP, and hTgs1 proteins as specified were incubated for 15 min at 37 °C. The extent of methyl transfer is plotted as a function of input enzyme.

FIGURE 10.

Effects of alanine mutations on hTgs1 methyltransferase activity. A, aliquots (1 μg) of the nickel-agarose preparations of wild-type hTgs1-(631–853) and the indicated hTgs1-(631–853)-Ala mutants were analyzed by SDS-PAGE. Polypeptides were visualized by staining the gel with Coomassie Brilliant Blue dye. The positions and sizes (kDa) of marker proteins are indicated on the left. B, methyltransferase reaction mixtures (20 μl) containing 50 μm [³H-CH₃]AdoMet, 1 mm m⁷GDP, and hTgs1 proteins as specified were incubated for 15 min at 37 °C. The extent of methyl transfer is plotted as a function of input enzyme. WT, wild type.

FIGURE 11.

Insights to Tgs1 from the structure of rRNA guanine-N² methyltransferase RsmC. Stereo view of the active site of T. thermophilus RsmC in complex with AdoMet and guanosine (from Protein Data Bank 3DMH). Highlighted are the RsmC side chains that contact the methyl donor and acceptor and that have putative counterparts in hTgs1. Hydrogen bond interactions are denoted by dashed lines. The residue numbers refer to the homologous positions in hTgs1.