Trimethylguanosine synthase (Tgs1) is the enzyme that converts standard m7G 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 tgs1Δ. 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 mud2Δ tgs1Δ and nam8Δ tgs1Δ 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.