Sequence–structure–function relationships of Tgs1, the yeast snRNA/snoRNA cap hypermethylase

ABSTRACTThe Saccharomyces cerevisiae Tgs1 methyltrans-ferase (MTase) is responsible for conversion of them 7 G caps of snRNAs and snoRNAs to a 2,2,7-trimethylguanosine structure. To learn more aboutthe evolutionary origin of Tgs1 and to identify struc-tural features required for its activity, we performeda structure–function study. By using sequence com-parison and phylogenetic analysis, we found thatTgs1 shows strongest similarity to Mj0882, a proteinrelated to a family comprised of bacterial rRNA:m 2 GMTases RsmC and RsmD. The structural informa-tion of Mj0882 was used to build a homology modelof Tgs1p which allowed us to predict the range ofthe minimal globular MTase domain and the local-ization of other residues that may be important forenzyme function. To further characterize functionaldomains of Tgs1, mutants were constructed andtested for their effects on cell viability, subcellularlocalization and binding to the small nuclear ribo-nucleoproteins (snRNPs) and small nucleolar RNPs(snoRNPs). We found that the N-terminal domain ofthe hypermethylase is dispensable for binding tothe common snRNPs and snoRNPs proteins butessential for correct nucleolar localization. Site-directed mutagenesis of Tgs1 allowed also the iden-tification of the residues likely to be involved in theformation of the m7G-binding site and the catalyticcenter.INTRODUCTIONSmall ribonucleoproteins (RNPs) are complexes required forprocessing RNA precursors into mature RNA species(reviewed in 1). Based on their intracellular location andfunction, these RNPs can be classified in two groups, thenucleoplasmic small nuclear RNPs (snRNPs) that play a rolein the maturation of pre-mRNAs and the small nucleolar RNPs(snoRNPs) that reside in the cell nucleolus and are required formaturation of pre-rRNA (reviewed in 2–4).The U1, U2, U4/U6 and U5 snRNPs are essentialcomponents of the spliceosome. They contain a set ofcommon proteins also called Sm proteins (B/B¢ in mammals,D1, D2, D3, E, F and G) that assemble as a heptamericdoughnut-like structure around the Sm site of the snRNAs (5).With the exception of U6, the snRNAs are transcribed by RNApolymerase II, acquire a m