Amino acid sequence conservation of Rmt1/Rmt3/PRMT3 and Rps2. (A) Aligned sequences for the N-terminal region of Rmt1 (S. cerevisiae, NP_009590), Rmt3 (S. pombe, NP_595572) and PRMT3 (M. musculus, NP_598501; H. sapiens, NP_005779). Identical residues are shaded, and the N-terminal zinc-finger motif is boxed in grey. (B) Aligned sequences for the N-terminus of Rps2 homologs, showing the RG-rich region (S. cerevisiae, NP_011392; S. pombe, NP_588435; M. musculus, NP_032529; H. sapiens, NP_002943). Protein arginine methyltransferase recognition motifs are shaded, including GRGG, RGG, and RXR [4, 34, 35].

Loss of methylation of Rps2 in Δrmt1 mutant cells. Intact cells were labeled with [³H]AdoMet as described in Materials and methods except that cells were grown initially in 1 l of medium and labeled with 2.0 nmol of [³H]AdoMet (Perkin Elmer Life Sciences, 83.3 Ci/mmol) in 40 ml of fresh YPD and the resulting cell pellets were lysed individually using 2 g beads and 4.5 ml buffer A for a total of ten cycles. Small ribosomal subunits were prepared by sucrose gradient centrifugation and the protein fraction isolated by ethanol/acetic acid treatment as described in Materials and methods. 50 μg of small subunit proteins were separated by SDS gel electrophoresis for 35 mA for 40 min and then 25 mA for 5 h with molecular weight standards (Bio-Rad Low Molecular Weight; hen ovalbumin, bovine carbonic anhydrase, and soybean trypsin inhibitor) in an adjoining lane. After protein staining (top panel), the gel was treated in EN³HANCE (Perkin Elmer Life Sciences) for 1 h, followed by a 20 min wash in water before being dried under vacuum for 2 h at 80° C followed by 1 h without heat. The gel was placed on Kodak BIOMAX XAR film for 3 months at -80° C (bottom panel). Large arrows indicate the position of the Rps2 band; small arrows indicate the position of the standards in the radioautograph.

Detection of asymmetric dimethylarginine and monomethylarginine residues in yeast Rps2. Amino acid analysis of the [³H]methylated ∼27 kDa polypeptide (Rps2) of the S. cerevisiae small ribosomal subunit. Cells (BY4742, RMT1+) were labeled in vivo with [³H]AdoMet and small ribosomal subunit proteins were isolated as described in Materials and methods. These proteins were fractionated by SDS gel electrophoresis as described in Materials and methods for 35 mA for 2.5 h, and then at 20 mA for 5 h. After Coomassie-staining, the band under the 31 kDa marker was excised, minced, and placed in a 6 × 50 mm flint glass tube. Acid hydrolysis and amino acid fractionation by ion exchange chromatography were performed as described in [16] with added standards of 1 μmol each of epsilon-dimethyllysine, N^G,N^G-dimethylarginine, and N^G-monomethylarginine. The open circles show radioactivity from 200 μl of 1 min (1 ml) fractions collected. The closed circles show the absorbance at 570 nm of ninhydrin assays of 100 μl of the fractions for the added methylated amino acid standards. In this high-resolution chromatography, the tritiated methylated derivatives elute slightly before the non-isotopically labeled standards [19].

Loss of methylation of Rps2 in yeast cells lacking the Rmt1 protein arginine methyltransferase. Small subunits were isolated from the ribosomal fraction of RMT1⁺ cells (wild type) and of Δrmt1^- cells prepared as described in Methods and materials but in 1 l of growth media without the [³H]AdoMet incubation. Due to greater cell pellet volumes, 4.5 ml buffer A was used to lyse each cell pellet and ribosomal pellets were individually re-suspended in 1 ml buffer B. An abbreviated gradient of 7%-27% sucrose made in buffer B was centrifuged for 16 h under the same conditions noted in Materials and methods. Small subunit proteins were prepared as described in Materials and methods except that dialysis was not performed and the sample supernatants after ethanol and acetic acid treatment were directly concentrated by vacuum centrifugation for about 35 min to bring the total volumes to approximately 100 μl. A final centrifugation of 20,000 × g for 5 min removed any particulate matter from solution, and 30 μl of each concentrated sample was analyzed by liquid chromatography/mass spectrometry using an Applied Biosystems Q-Star Elite instrument as described in Materials and methods. Data for the peak at 51 min containing Rps2 were de-convoluted using Analyst software and the resulting average masses of significant peaks are shown. Mass changes are indicated for 14 Da (methylation) as well as 16 Da (oxidation).