CHARACTERIZATION OF A NUCLEOLAR 2'-O-METHYLTRANSFERASE AND ITS INVOLVEMENT IN THE METHYLATION OF MOUSE PRECURSOR RIBOSOMAL-RNA

A nucleolar 2'-O-methyltransferase, partially purified from isolated mouse nucleoli, catalyzes the methylation of each of the four nucleosides, although to different levels depending on the RNA substrate. Similar to most methyltransferases which use S-adenosyl-L-methionine (SAM) as the methyl donor, the nucleolar 2'-O-methyltransferase was shown to bind S-adenosyl-L-homocy-steine (SAH) (K-d = 0.17 mu M), a product of the transfer reaction, as tightly as SAM (K-d = 0.24 mu M). Binding assays also demonstrated stereospecificity about the sulfonium center of SAM. The naturally occurring S-chiral form of SAM had a 10-fold higher binding affinity than the R-chiral form. In addition, the alpha-amino group of the methionine moiety and the 6-amino group of the adenine moiety were shown to be required for maximal binding. The relative high affinity for both SAM and SAH may reflect a mechanism by which ribosome biogenesis is, in part, coordinated with cell growth, since a decrease in SAM:SAH ratio correlates with decreasing levels of 2'-O-methylation. The availability of unmethylated, in vitro-derived rRNA transcripts has made it possible to explore questions relating to the specificity for the RNA substrate. Using an in vitro-derived 28S rRNA transcript, the enzyme selectively methylated the sequence AmGmCm that occurs in a single-stranded bridge spanning two highly conserved structural domains of 28S rRNA. These results demonstrated that the purified nucleolar 2'-O-methyltransferase was sufficient to accurately methylate this region of 28S rRNA, and were taken to support the involvement of this nucleolar enzyme in the posttranscriptional methylation of the 47S precursor ribosomal RNA transcript. To identify the protein species associated with the 2'-O-methyltransferase, the enzyme was photoaffinity labeled using a 28S rRNA transcript synthesized to contain the photoreactive uridine analog, 5-azidouridine. Bound nucleolar 2'-O-methyltransferase was irradiated with UV light, and following ribonuclease digestion to destroy the RNA component of the complex, labeled material was separated either under denaturing conditions on a SDS-polyacrylamide gel, or native conditions by molecular sieving column. On SDS-polyacrylamide gel, a specifically labeled polypeptide band was observed at a size of similar to 50 M(r). In contrast, under the native conditions of a molecular sieving column, photolabeled enzyme eluted at a position for a protein of similar to 150 M(r), consistent with the size estimated for the 2'-O-methyltransferase activity and SAM binding activity. These results suggest that the nucleolar 2'-O-methyltransferase has quaternary structure. Since the average size for most methyltransferase activities ranges from similar to 30 to similar to 60 M(r), the increased size and apparent complexity for the nucleolar 2'-O-methyltransferase may be important in the regulation and specificity of the enzyme.