Volume 59, number 1 FEBS LETTERS November 1975 TISSUE SPECIFIC DIFFERENCES IN THE 2'-O-METHYLATION OF EUKARYOTIC 5.8S RIBOSOMAL RNA Ross N. NAZAR, Thomas O. SITZ and Harris BUSCH Tnmor By-Products Laboratory. Department o f Pharmacology, Baylor College of Medicine, Houston, Texas 77025, USA Received 11 August 1975 1. Introduction Although a great deal is known about protein syn- thesis, the means by which ribosomes carry out their nmltiple functions and the mechanisms for their con- trol are not defined. The role of modified nucleotides, for example, is essentially unknown. About 3--4% of the nucleotides in eukaryotic ribosomal RNA are modified; 1-2% of the ribose residues are 2'-0- methylated and 10% of the uridylic acid residues are converted to pseudouridylic acid [1,2]. Some of these modifications are present in the entire RNA population while others are restricted to a fraction of the molecules [3,4]. Methylation appears to be essen- tial in ribosome maturation [5] and ribose-methyl- ation is largely or completely restricted to the mature RNA species [6]. It has been suggested that methyl- ation may render crucial sequences resistant to cyclizing ribonucleases [7]. Alternately, the presence of partially methylated or modified sequences has suggested that they may exert some modulating or control activity. Recent studies on the primary sequence of Novikoff ascites hepatoma 5.8S rRNA have revealed two sequences containing 2'-O-methylribose, A-A- U-U-Gm-C-A-Gp and G-G-Um-G-G-A-Up and two sequences containing pseudouridylic acid, C-ff-Gp and ff-Gp [8,9]. The 2'-O-methyl guanylic acid residue was present in molar amounts while Um was only found in about 20% of the molecules. Simi- larly, the ff-Gp sequence was present in every mole- cule while C-ff-Gp was only found in half molar amounts. An experimental approach to the role of modified nucleotides in ribosomal RNA requires an ability to alter the levels of modification for correla- tion with its biological function. As a prelimin~lry approach towards understanding the role of these modified nucleotides, we have examined 5.8S rRNAs from various tissues of different growth rate and development in search of physiologically related changes. 2. Materials and methods The tissues used in these studies were human, rat, mouse or chick in origin. Normal BALB/C mice and the transplanted DMBA(dimethylbenzanthracene)- induced mouse mammary tumors were kindly provided by Dr D. Medina. Mouse myeloma MPC-1 1 cell RNA, HeLa cells and secondary chick embryo cells were pro- vided by Drs E. Murphy, P. N. Rao and J. Norris, respec- tively. Male Sprague Dawley rats were used for nor- mal rat tissues and to maintain Novikoff hepatoma ascites cells. Cells were labeled with 5-50 mC: of [32P]orthophosphate in vitro or in vivo as previously described [ 10] ; for regenerating liver the isotope was injected interperitoneally 48 h after the partial hepatectomy and the animal was sacrificed 24 h later. Whole cell RNA was extracted with a phenol-SDS buffer at 65°C and the 5.8S rRNA waKfractionated on polyacrylamide gel slabs [8,9]. The purified RNA was digested with pancreatic or TI ribonucleases and the resulting oligonucleotides were fractionated by two-dimensional electrophoresis [ 11 ]. The modified fragments were identified as previously reported [8]. North-Holland Publishing Company - Amsterdam 83