High molecular weight nonribosomal-type nuclear RNA and cytoplasmic messenger RNA in HeLa cells.

As reported elsewhere,' the study of RNA synthesis in vitro in immature duck erythrocytes has revealed the occurrence in the nucleus of fast turning-over RNA of high molecular weight (from 2 X 106 to 107, or possibly more) and with base composition different from that of ribosomal RNA, and characterized by a high U and relatively low GC content. No relationship of precursor-to-product typeor, at any rate, not a simple one-appears to exist between this RNA and the messenger RNA fraction associated with cytoplasmic polysomes. In this paper, evidence is presented indicating that this class of nuclear RNA molecules is not exclusive of immature erythrocytes, or in general of nondividing cells undergoing differentiation, but occurs also in exponentially growing cells. Materials and Methods.-(a) Cells: HeLa cells growing in suspension in a modified Eagle's medium2 supplemented with 5% calf or horse serum were utilized throughout. The cultures used here were free of any detectable contamination with PPLO (Mycoplasma). (b) Labeling conditions: Exponentially growing HeLa cells, at a concentration of 1-3 X 105 cells/ml, were exposed for various times to H3-uridine (0.01 mM, 0.2-0.3 mc/MM) or C14-uridine (0.01 mM, 5 jAc/IAM). In the experiments involving the use of P32-orthophosphate, the cells were washed twice in phosphate-free Eagle's medium (with dialyzed serum) and then resuspended in the same medium, supplemented, in most cases, with 10-5 M phosphate; carrier-free P32-orthophosphate was utilized at a concentration of 20-30 ttc/ml. Long-term labeling of HeLa cell protein was carried out by growing the cells for 48 hr in leucineand lysine-free Eagle's medium (+5% dialyzed serum) supplemented with L-H3-leucine (1.25 ,4c/ml; 16.7 ,4c/,4M) and L-H3-lysine (0.63 Ac/ml; 8.3 Muc/,.M). (c) Isolation of polysomes: The labeled cells to be utilized for polysome extraction were washed three times with 0.13 M NaCl, 0.005 M KCl, 0.0075 M MgCl2, resuspended in 6 vol of 0.01 M tris buffer, pH 7.4, 0.01 M KCl, and 0.0015 M MgCl2, left 5 min at 0-20C, then homogenized with an A. H. Thomas homogenizer. The homogenate was centrifuged for 10 min at 600 X g at 20C to separate the nuclear from the cytoplasmic fraction; ribosomes and polysomes were isolated from the latter by a method previously described. ' (d) Extraction and analysis of RNA: Total RNA was routinely isolated by a procedure involving cold phenol-sodium dodecylsulfate extraction of total nucleic acids followed by digestion of DNA by RNase-free DNase.' Conditions forRNA extraction from polysomes, sedimentation analysis of RNA, enzymatic tests, isotope determinations, and base composition analysis are described in detail elsewhere.' Results.-(a) Sedimentation pattern of rapidly labeled RNA after different times of exposure of the cells to a labeled RNA precursor: After a short exposure (up to 30 min) Eo H3or C'4-uridine or P32-orthophosphate, only a small proportion of the total radioactivity incorporated into RNA is found to be associated with the cytoplasmic fraction, under conditions of cell breakage which release into this fraction 50-70 per cent of the total RNA. This result confirms previous findings in this and other animal cell types,3-6 indicating a nuclear localization of the earliest-labeled RNA components. An analysis of the sedimentation behavior of this fast-labeled RNA shows that, after a 5-min pulse, the labeled components are distributed in the region of the sucrose gradient corresponding to S values of about 10 to 100 or possibly more, with a greater accumulation in the 30-65S region (see, e.g., top graph of Fig. 4).