Volume 9, Issue 4, Pages 891-901 (April 2002) Hypermethylation of the Cap Structure of Both Yeast snRNAs and snoRNAs Requires a Conserved Methyltransferase that Is Localized to the Nucleolus  John Mouaikel, Céline Verheggen, Edouard Bertrand, Jamal Tazi, Rémy Bordonné  Molecular Cell  Volume 9, Issue 4, Pages 891-901 (April 2002) DOI: 10.1016/S1097-2765(02)00484-7

Figure 1 In Vitro Binding of Tgs1p [35S]methionine-labeled Tgs1p, prepared by in vitro transcription and translation, was mixed with the indicated GST fusion proteins produced in E. coli. Proteins bound to glutathione-Sepharose beads were washed, denatured, and separated by SDS-PAGE. Input represents aliquots of radioactive proteins corresponding to 20% of that used in each of the binding reactions. The additional low migrating band corresponds to a premature translation termination product of Tgs1p. The Sm domains used as GST fusions in (B) and (C) are schematically shown in (A). Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)

Figure 2 Evolutionary Conservation of Yeast Tgs1p (A) Schematic representation of Tgs1p and its relatives. The lengths (amino acid) of the proteins are shown at the right. The conserved sequences are shaded. The A. thaliana protein presents a duplication of this region. The N-terminal domain of X. laevis and the C-terminal region of the T. brucei proteins are not complete. (B) Diagrammatic representation of the motifs I and post I in relation to S-AdoMet binding. The conserved motifs of the yeast sequence are shown in the model proposed in Niewmierzycka and Clarke (1999). The black arrowheads indicate residues D103 and D126 that were mutated to alanine to prevent AdoMet binding. Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)

Figure 3 Properties of the tgs1::KAN Deleted Strain (A) Cold-sensitive phenotype of the tgs1::KAN strain. Exponentially growing cultures of yeast cells were serially diluted, spotted on plates, and incubated at 16°C or 28°C. In contrast to the corresponding wild-type strain, the tgs1::KAN strain was unable to grow at 16°C but grew when transformed with a plasmid carrying a GFP-Tgs1 fusion. Mutations in the AdoMet binding site inhibited Tgs1p activity as shown by the inability of the GFP-Tgs1/D103A and GFP-Tgs1/D126A mutant fusion proteins to complement the cold-sensitive phenotype of the tgs1::KAN strain. (B) Coimmunoprecipitation. Whole-cell extracts prepared from the indicated yeast strains were immunoprecipitated with anti-GFP antibodies absorbed by protein A-Sepharose beads. The proteins present in the pellet (P.) and the supernatant (S.) were subjected to Western blot analysis. The additional bands found in the pellets arise from the protein A-Sepharose compound. (C) Cold-sensitive inhibition of pre-U3 splicing in tgs1::KAN cells. After growth of wild-type cells and cells carrying tgs1::KAN or prp4 alleles at the indicated temperature, total RNA was isolated and used for primer extension. Arrows indicate the two species corresponding to the U3 RNA precursors. (D) Glycerol gradient sedimentation of yeast snRNPs. Splicing extracts prepared from the indicated strains were incubated with ATP and subjected to glycerol gradient sedimentation. The RNAs were then transferred to a nylon membrane for Northern blot analysis using probes specific for U4, U5, and U6 snRNAs. The fraction number of each gradient and the positions of the different snRNP complexes are shown at the bottom. Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)

Figure 4 Tgs1p Is Required for Hypermethylation of snRNAs (A) Whole-cell extracts were prepared from wild-type and tgs1::KAN strains grown at 28°C, and snRNPs were immunoprecipitated with anti-m3G antibodies specifically recognizing the m3G cap. RNA was extracted from the supernatants (S.), from the pellets (P.), and from equivalent aliquots of the total lysates (Total), separated on denaturing polyacrylamide gels, and subjected to Northern analysis. Hybridization was performed with probes specific for the yeast U1, U2, U4, U5, and U6 snRNAs. (B) Immunoprecipitations of snRNPs were performed on whole-cell extracts prepared from the indicated strains as described in (A), except that anti-H20 antibodies recognizing both m3G and m7G caps of snRNAs were used. Northern analysis was done with probes specific for U4, U5, and U6 snRNAs. (C) Expression of a GFP-Tgs1 fusion protein restores m3G cap formation while mutations in the AdoMet binding site of Tgs1p inhibit m3G cap formation. Whole-cell extracts prepared from tgs1::KAN cells expressing the indicated GFP fusion proteins were grown at 28°C and used in immunoprecipiation experiments as described in (A). Hybridization was done with probes specific for U4, U5, and U6 snRNAs. Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)

Figure 5 Tgs1p Is Required for Hypermethylation of snoRNAs and Interacts with the Basic Repeats in Nop58p and Cbf5p (A) Whole-cell extracts were prepared from wild-type and tgs1::KAN strains grown at 28°C, and snoRNPs were immunoprecipitated with anti-m3G antibodies specifically recognizing the m3G cap and treated for Northern analysis as described in the legend of Figure 4A. Hybridization was performed with probes specific for the indicated yeast snoRNAs. (B) In vitro binding assays. [35S]methionine-Tgs1p prepared by in vitro transcription and translation was mixed with the indicated GST fusion proteins produced in E. coli. Proteins bound to glutathione-Sepharose beads were treated as described in the legend of Figure 1. Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)

Figure 6 Nucleolar Retention of U1 snRNA in tgs1 Mutant Strains (A) Wild-type, tgs1::KAN, and tgs1::KAN+Tgs1/D103A cells carrying a GFP-Gar1 construct were grown at 20°C, and the endogenous U1 snRNA (red) was detected by fluorescent in situ hybridization. The nucleolus was labeled with a GFP-Gar1 fusion protein (green), and the DNA was stained with DAPI (blue). (B) The indicated strains were grown and treated as in (A), except that fluorescent in situ hybridization was done with a U6 antisense probe. Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)

Figure 7 Tgs1p Localizes to the Nucleolus Exponentially growing wild-type cells carrying the indicated GFP fusions were grown in liquid media at 28°C. U3 snoRNA (red) was detected by fluorescent in situ hybridization and the indicated GFP fusion proteins (green) were detected by immunofluorescence. The DNA was stained with DAPI (blue). Molecular Cell 2002 9, 891-901DOI: (10.1016/S1097-2765(02)00484-7)