Abstract: Trimethylguanosine synthase (Tgs1) is the enzyme that converts standard m7G caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal snRNAs. 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 tgs1. The Tgs1 interaction network embraced proteins implicated in snRNP function and spliceosome assembly, including Mud2, Nam8, Brr1, Lea1, Ist3, Isy1, Cwc21 and Bud13. Complementation of the synthetic lethality of mud2 tgs1 and nam8 tgs1 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 Pol II transcription (Rpn4, Spt3, Srb2, Soh1, Swr1, Htz1). We find that the C-terminal domain of human Tgs1 (hTgs1) can function in lieu of the yeast protein in vivo. We present a biochemical characterization of the hTgs1 guanine-N2 methyltransferase reaction and identify individual amino acids required for methyltransferase activity in vitro and in vivo.