Nascent ribosomes from HeLa cells.

Abstract

In recent years several stages in the synthesis of mammalian ribosomes have been resolved. (a) The 45S precursor RNA is synthesized in the nucleolus as a single polynucleotide chain.' (b) The 45S RNA is cleaved in a number of steps to 32SRNA and 18S RNA.1-3 (c) While still in the nucleolus, the 32SRNA is further cleaved to 28S RNA,3 to which is bonded noncovalently a small, 7S, piece of RNA.4 7S RNA may be released by heat, dimethyl sulfoxide, or urea. (d) The mature 28S and 18S RNA's can be detected transiently in ribonucleoprotein particles in the nucleus.5 (e) The individual subribosomal particles pass quickly to the cytoplasm where they become incorporated into polyribosomes and subsequently equilibrate with single ribosomes.6 7 A major point of interest is the process by which ribosomal protein is assembled with ribosomal RNA, which probably occurs in the nucleolus. Since available techniques for nuclear fractionation have not yet permitted a direct approach to this problem, we have attempted to isolate from the nucleolus ribonucleoprotein particles in various stages of development toward cytoplasmic ribosomes. Two types of such particles have been identified, which we have termed ribosomal for reasons which will become evident. These particles may be similar to those described by Tamaoki and Mueller,8 and by Yoshikawa-Fukada.9 Materials and Methods.-Cells: HeLa S3 cells were grown in spinner culture in Eagle's'0 minimal essential medium (MIEM) supplemented with 7% horse serum. Solutions: RSB: 0.01 M NaCl; 0.0015 Mll MgCl2; 0.01 M Tris, pH 7.4; HSB: 0.5 M NaCl; 0.05 M MgCl2; 0.01 M Tris, pH 7.4; NEB: 0.01 M NaCl; 0.01 M EDTA; 0.01 M Tris, pH 7.4; NETS: 0.1 M NaCi; 0.001 M EDTA; 0.01 AM Tris, pH 7.4; 0.1% SDS. Radioactive labeling of cells: Cells were labeled with C14and H3-uridine as previously desCribed.6 For labeling with C'4-leucine, a culture was centrifuged at 1500 rpm for 2 min, and the resulting cell pellet was resuspended in 1/' to 1/lo the original volume of a modified Eagle's MEM, containing 7% whole serum but no added leucine. C'4-leucine was added for a given length of time, followed by dilution of the culture to the original volume in Eagle's complete MEM with serum plus ten times the normal concentration of leucilne. Cell fractionation: Nutcleoli were prepared by a modification of the method of Penman.2 Cells from a 100-ml culture were harvested by centrifugation at 1500 rpm for two min, followed by two washes with cold Earle's solution. All subsequent steps were carried out at 4°. The cells were resuspended in 1.5 ml RSB and were subsequeiitly ruptured with a I)ounce homogenizer. The nuclei were collected by centrifugation for 3 mim at 1300 rpm and were washed with another 1.5 ml RSB. Ribosomes were prepared from the pooled supernatants by Mg++ precipitation as previously described.'2 The nuclei were suspended in 2.0 ml RSB to which was added 0.3 ml of a solution containing 6.7% Tweeni 40 and 3.3% sodium deoxycholate. The suspension was briefly mixed on a Vortex mixer and centrifuged at 1500 rpm for 5 min. The gelatinous pellet of nuclei was resuspended in 2.5 ml of cold HSB containing 100 lOg D)Nase (Worthington, electrophoretically). The viscosity of this suspension was reduced markedly after vigorous pipetting for several minutes. The crude nucleoli were collected by centrifugation in the cold for 10 min at 10,000 rpm. The nucleolar pellet was resuspended in 1.5 ml of NEB containing 0.01 M dithiothreitol and gently stirred for 15 min at room temperature. After centrifugation for 10 min at 15,000 rpm the supernatant, containing the nascent ribosomal particles, was analyzed on a 15-30% w/w sucrose gradient in NEB solution.