Polyadenylate sequences have been found covalently linked in heterogeneous DNA-like nuclear RNA of HeLa cells. This poly(A) material seems homogeneous in size and accounts for about 0.5% of such RNA. Similar poly(A) sequences were found in rapidlylabeled polyribosomal RNA, thought to be messenger RNA. A possible model for mRNA synthesis from large heterogeneous nuclear RNA precursor molecules is discussed.The synthesis of a relatively homogeneous population of polyriboadenylic acid [poly (A) I sequences of 150-200 nucleotides in the nucleus of Ehrlich ascites cells was recently described (1). The suppression of this synthesis with actinomycin D suggested that AMP-rich sequences were transcribed on DNA. Knowledge of the precise location of these sequences with respect to those RNA species which characterize eukaryotic cells was required to interpret these observations. Suspension cultures of HeLa cells were selected for such studies, not only for the techniques available for the isolation of wellcharacterized subcellular components (2) and RNA species (3, 4), but particularly for the detailed knowledge, available from kinetic analyses, of the flow of radioactivity through and into various RNA species of these cells (5).The poly(A) content of RNA fractions from the nucleus and cytoplasm of HeLa cells has been measured by a technique dependent on the hydridization of poly(A) sequences to polydeoxythymidylate oligomers covalently bound to cellulose (6). Highly purified poly(A) can be quantitatively recovered from suitably prepared RNA samples on temperaturecontrolled filters of poly(dT)-cellulose (7).We show here a broad distribution of poly(A) sequences within two classes of RNA that are themselves characteristically heterogeneous, i.e. the rapidly labeled polydisperse RNA (HnRNA) of the nucleus, and the polyribosome-associated RNA with the sedimentation characteristics of messenger RNA (mRNA). The poly(A) sequences recovered from all size classes of both types of RNA are rather homogeneous and show similar electrophoretic mobilities in acrylamide gel that correspond to an approximate length of 150-200 nucleotides.Evidence is presented that the poly(A) sequences detected after ribonuclease digestion of the HnRNA are in fact covalently bonded within that RNA. Equally compelling evidence for the covalent linkage of poly(A) detected in mRNA to that RNA has not yet been obtained, although such a linkage is considered likely.The implications of the detection of poly(A) sequences of similar length in both cytoplasmic mRNA and nuclear HnRNA will be considered with respect to a precursorproduct relationship proposed (8) to link these RNA species. MATERIALS AND METHODS Cell cultureSuspension cultures of HeLa cells were maintained as previously described (9), at cell concentrations of 2-5 X 105 cells/ml. Cell labeling and fractionationCells were concentrated to 3 X 106 cells/ml for labeling with 32PO4 (0.2 mCi/ml) in phosphate-free Eagle's minimal essential medium, supplemented with 5% normal calf serum. After the la...

The subcellular distribution of various types of RNA in HeLa cells is described. In addition, the relative rate of synthesis of the major classes of nuclear RNA has been determined. From these experiments it can be deduced that the heterogeneous nuclear RNA fraction is rapidly synthesized and degraded within the cell nucleus.

MIany aspects of ribosomal RNA synthesis and ribosome formation in cultured animal cells have been clarified over the past several years. Ribosomal precursor RNA synthesis in the cell nucleolus' 2 is rapidly followed by methylation of the RNA.', 4 Within 20-40 minutes of synthesis this new RNA can be detected in nuclear subribosomal particles which are precursors tocytoplasmicsubribosomal particles.' It thus becomes possible to study in detail the effects of various metabolic restrictions on ribosome formation.The present experiments are concerned with the effects of methionine starvation. In HeLa cells, methionine not only is an essential amino acid for the formation of protein but also furnishes methyl groups in the methylation of nucleic acid bases and of the 2'-OH group of ribose, the major site of methylation in HeLa ribosomal RNA.'7As will be shown, in the absence of exogenous methionine, complete ribosomes are not formed. Evidence is presented that the degree of methylation of ribosomal precursor RNA is severely limited, and that this may be the factor limiting ribosomal maturation. In spite of this restriction of ribosome completion, the transcription of ribosomal precursor RNA continues. In contrast, cells deprived of valine, another growth-essential amino acid without any special function in the formation of ribosomal RNA, continue to initiate ribosomal RNA synthesis and to complete ribosomes for many hours. This is presumably due to the provision of valine via protein turnover.8 Materials and Methods.-Suspension cultures of HeLa cells (3-6 X 105 cells/ml) growing in Eagle's medium9 were starved for methionine by centrifuging cells and resuspending them, at 370, for 6 hr in Eagle's medium lacking methionine (or valine), but supplemented with 7% dialyzed horse serum. Vraline deprivation was accomplished by 15 hr incubation in medium lacking valine but supplemented with 7% normal serum. Starved and control growing cultures were labeled with C'4-uridine or -adenine (both 30 4c/humole, New England Nuclear Corp.) at 0.15 ,gc/ml for 2.5 hr (CL4-uridine) or 6 hr (C'4-adenine). Growing cells were labeled with C14-methyllabeled methionine by first growing cells in the presence of 10-4 M adenosine for 24 hr, then removing the cells from growth medium and resuspending them in methionine-free medium to which adenosine and CL4-methyl-labeled methionine (0.4 ,c/ml, 14 Lc/smole) were added. The inclusion of adenosine prevents the use of -CH3 groups of the methionine in synthesis of purines. AMethionine-starvel cells to be labeled with p32 were centrifuged from the starvation medium after 6 hr, resuspended and incubated for an additional 6 hr in medium lacking the normal P04 as well as methionine but containing dialyzed serum and 0.13 mc/ml of carrier-free P3204.RNA extraction and sedimentation analysis: Cells were fractionated into cytoplasm, detergent cleaned nuclei, nuclear supernatanit fraction, and nuclear pellet (tuicleolar) fraction by methods previously described ill detail."0 "1 The RNA of cytoplasmic extracts...

Poliovirus virion RNA contains a single covalently bound sequence of polyadenylic acid which is approximately 49 nucleotides long. A single, slightly longer polyadenylic acid sequence is contained in Eastern Equine Encephalitis virus RNA. Since these viruses are otherwise dissimilar these sequences may play a common role in viral replication, possibly in translation of the viral RNA.