Acrylamide gel electrophoresis of HeLa cell nucleolar RNA.

Abstract

The nucleolus of eucaryotic cells has been identified by Perry and others'-4 as the site of ribosomal RNA synthesis. It has recently become possible to obtain from HeLa cells a nucleolar preparation which, as seen by electron microscopy, is relatively free of chromatin.5' I Fractionation of C'4-uridine-labeled cells confirmed the hypothesis that the nucleolus is the site of synthesis of the 45S ribosomal RNA precursor.5 Another species of RNA (32S) is present in relatively large amounts. The nucleolus appears to contain only ribosomal precursor RNA since if fractionation is performed carefully, very little of the nucleoplasmic heterodisperse RNA is associated with it.7 8 The following picture of the major events in ribosomal RNA formation has emerged. The initial event is the synthesis of a high-molecularweight precursor molecule with a sedimentation constant of 45S.Y-1 After 15 to 20 minutes, this molecule is cleaved, yielding 18S ribosomal RNA and a species of RNA whose sedimentation constant is 32S.12 The 18S RNA is quickly transported from the nucleolus and appears in the cytoplasm as part of the smaller ribosomal subunit. After additional processing time in the nucleolus, the 32S molecule is converted to 28S and eventually emerges into the cytoplasm as part of the larger ribosomal subunit. With the development by Loening of a method of polyacrylamide gel electrophoresis for molecules as large as ribosomal RNA, 3 it has become possible to study in greater detail the events of nucleolar RNA processing. The gels used in these experiments have been modified by the addition of glycerol to facilitate freezing and slicing. The following information has been obtained concerning nucleolar processing of ribosomal RNA. (1) The site of transformation of 32S to 28S RNA is the nucleolus. (2) Several additional short-lived intermediate species of ribosomal RNA have been identified with estimated sedimentation constants of 41S, 36S, and 20S. (3) Some short-lived intermediates increase in amount under conditions that disrupt normal nucleolar RNA processing, e.g., poliovirus infection. (4) The conversion of 45S RNA to mature ribosomal RNA is accompanied by a net loss of RNA. This is also shown for the transformation of 32S RNA to 28S. Materials and Methods.-Cells: HeLa type 3 cells were grown and labeled in suspension culture as previously described.14 Radioisotopes: Imethionine-methyl-C'4 (49 mc/mM) and uridine-2-C'4 (27 mc/mM) were purchased from Schwarz BioResearch. Imethionine-methyl-H3 (1400 me/mM) was purchased from Nuclear Chicago. Methyl labeling was performed in Eagle's medium free of unlabeled methionine and containing adenosine and guanosine (2 X 10-5 M). When noted, unlabeled methionine was added back to this medium to prevent methionine starvation. Cell fractionation: Cells were separated into nuclear and cytoplasmic fractions as previously described.1' The cleaned nuclei from approximately 4 X 107 cells were suspended in 1 ml of highionic-strength buffer (HSB: 0.5 M NaCl, 0.05 M MgCl2, 0.01 M Tris, pH 7.4), warmed briefly to 370, and digested with 50 yg of Worthington electrophoretically purified DNase. The digest