Vol. Ribonucleic Acids from Animal Cells · incorporated radioactivity (i.e., newly made RNA)is notin themajorityspecies ofRNA(85). Figure 3 shows the sedimentation pattern of the

BACTERIOLOGICAL REVtEWS, Sept., 1968, p. 262-290 Vol. 32, No. 3Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Ribonucleic Acids from Animal CellsJAMES E. DARNELL, JR.1

Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, Yeshiva University,New York, New York 10461

INTRODUCTION ................................................................ 262TECHNICAL ASPECTS OF RNA BIOCHEMISTRYC.. .. 263

Size ...263Compositio.... . 263Methylation ...263RNA-DNA Hybridization ...264Cell Fractionation ........................................................... 264

PATTERNS OF SYNTHESIS OF ANIMAL CELL R.. ........ .. 264"Rapidly Labeled RNA" and Kinetic Analysis Experimes .... 264

BIOSYNTHESIS OF RlBOSOMES IN ANIMAL CELLS .... 267Ribosomal PrecursorRNA..... 267Formation and Distribution of Ribosomal Particles .. 272Ribosomal Proteins and Nucleolar Ribosomal Precursors 274Association of Ribosomal Protein withrRNA.. 276Place of Small RNA Molecules in RibosomalMaturatiot.. 276Control of Ribosome Formation.277

PRECURSORS TO TRNA MOLECULES....279DNA-LIKE RNA SPECIES...................................................... 280

Definition and Distribution of DNA-like RNA.. 280HnRNA: Properties and Problems.283Use ofRNA-DNA Hybridizationz in the Study ofDNA-like RNA Species.284

CONCLUSIONS ............................................. 286LITERATURE CITED ........................................................... 287

INTRODUCTION

One of the important branches of molecularbiology at the present time is the study of thesynthesis, structure, and function of ribonucleicacid (RNA) molecules; yet only within the past15 years has it been generally appreciated thatcellular RNA comprises not a uniform collectionof polymers but many different molecules ofdefined length. The first distinguishable class ofRNA molecules was transfer RNA (tRNA), thecarrier of amino acids for protein synthesis (44,114). Another signal event in the development ofsuccessful studies on RNA molecules was thedemonstration that the RNA of tobacco mosaicvirus (TMV) was infectious by itself (32) andconsisted of a very long polymer, about 6,000nucleotides (30). Furthermore, a damagingchemical or physical event anywhere along thevirus RNA chain resulted in inactivation of in-fectivity of the RNA, implying the necessity forintegrity of the whole molecule for viral replica-tion (62).

It was soon found that many RNA-containinganimal viruses also possessed infectious RNA

1 Career Scientist of the Health Research Councilof the City of New York.

molecules comparable in size to TMV (84). Thesestudies on viral RNA were important to the de-velopment of RNA biochemistry in general be-cause they provided a yardstick by which otherRNA preparations could be judged. Thus, hope-fully, extraction procedures which yielded wholeviral RNA molecules from infected cells (108)could be used to extract cellular RNA in anundegraded state.The greatest impetus in recent times to the

study of RNA metabolism was provided by theprediction of messenger RNA (mRNA) in 1960-1961 (51), followed shortly by its experimentaldemonstration in bacteria (9, 38). This hithertounrecognized RNA species was the direct bearerof genetic information between deoxyribonucleicacid (DNA) and the protein-synthesizing appa-ratus. These events made the examination ofRNAin animal cells a pressing experimental necessity.Cultured animal cells offered a source of virtuallyunlimited numbers of clonally derived cells whichcould be easily labeled with radioisotopes. There-fore, experiments were begun which aimed atidentifying various classes of animal-cell RNAto compare with those identified in bacteria.From cytochemical and radioautographic data,virtually all (excluding mitochondrial DNA) of

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RIBONUCLEIC ACID FROM ANIMAL CELLS

the cellular DNA was known to be nuclear, andmost of the RNA and protein synthesis wasknown to be cytoplasmic (36, 73, 77, 97). Thus,it might have been anticipated that much of thework of RNA isolation and characterization inanimal cells would involve nuclear RNA speciesand their possible relationship to the cytoplasmicRNA species. This has indeed proved to be thecase, and the problems attendant to this questionhave by no means been completely solved. Enoughis now known, however, especially about ribo-some biosynthesis, to justify a general summaryof RNA synthesis in cultured animal cells. Al-though most of the experiments to be describedwere performed in cultured cells, much of thefuture interest will center on the role of RNA infunctioning cells, differentiated cells, and, es-pecially, in differentiating cells. Reference willtherefore also be made to specific experimentsdealing with differentiated cells. The author takesthe prerogative, however, of not attempting tocover comprehensively all points dealing withRNA synthesis in differentiated or, for thatmatter, cultured cells.

TECHNICAL ASPECTS OF RNA BIOCHEMISTRY

Before dealing with experimental results, itwill be helpful to review briefly the major tech-niques involved in examining RNA molecules.A completely satisfactory comparison of oneRNA species with another ultimately demands aknowledge of the linear arrangement of nucleo-tides in the two species. Recent years have seenremarkable advances along these lines, so thatthe sequence of a number of tRNA moleculesfrom yeast and Escherichia coli (46, 60) and of the5S ribosomal RNA (rRNA) molecules from E.coli (15) and KB cells (27), a strain of culturedhuman carcinoma cells, have been determined.These molecules, however, range between 75 and121 nucleotides in length. The time when com-plete sequences of longer polynucleotides will beavailable is considerably in the future. It is neces-sary, therefore, to turn to less decisive techniquesfor comparing the physicochemical properties ofvarious RNA molecules.

SizeSedimentation velocity is the most common

property determined on an isolated RNA sample.Although very accurate conclusions about themolecular weight of various RNA species cannotbe obtained by sedimentation velocity experi-ments (e.g., the sedimentation rates of differenttypes of RNA respond differently to changes inionic strength of the suspending medium), it ispossible by zonal sedimentation to assess the

range of molecular weights and the homogeneityof an RNA sample. Sucrose gradient zonalsedimentation has therefore become a standardtechnique for analyzing and preparing RNA (10).A recent addition to the techniques which sepa-rate single-stranded RNA molecules largely onthe basis of chain length is acrylamide gel elec-trophoresis of RNA (59). This technique offerstechnical advantages of speed and superior resolu-tion between RNA species of very similar sizes,but for preparative purposes the density gradienttechniques are still more widely employed.

CompositionAfter the separation ofRNA with a homogene-

ous sedimentation behavior, the overall composi-tion of the sample with respect to the fourcommon ribonucleotides (base composition)can be determined. The technique usually em-ployed is alkaline hydrolysis, followed by separa-tion of the resulting 2', 3' ribonucleotides bycolumn chromatography or by paper electro-phoresis (18, 83). If a sufficiently large RNAsample is available, the amounts of the fourribonucleotides can be estimated by ultraviolet(UV) absorption. Frequently, however, only asmall amount of a radioactive RNA species isavailable, and it may even contain a large amountof another type of nonradioactive RNA. In thiscase, if the radioactive species is labeled with32p, the base composition can be determined byassaying the distribution of 32p among the fourribonucleotides released by alkaline hydrolysis.This latter method, in comparison with UVanalysis of large amounts of purified RNA, hasproven quite satisfactory for the determinationof base composition of long polynucleotides,even after brief periods of label (83, 90).A simple technique has been developed over

the past several years which carries the analysisof RNA molecules one step further than simpleaverage base composition. In place of alkalinehydrolysis, which produces 2', 3' mononucleo-tides, the RNA can be subjected to controlledenzymatic digestion with pancreatic ribonucleaseor Ti ribonuclease (an enzyme from Aspergillusoryzae which preferentially cleaves RNA chainson the 3' side of guanylic acid residues). Thisresults in a specific pattern of oligonucleotidesfrom each type of RNA molecule. Even if differ-ent RNA molecules have identical overall basecomposition, their oligonucleotide patterns canbe distinguished (94).

MethylationAs will be discussed in more detail later, the

rRNA and tRNA molecules of animal cells,

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just as was first shown to be the case in bacterialcells, have methyl groups transferred to themat the polynucleotide stage from the terminalmethyl group of methionine. Other types ofcellular RNA lack these methyl groups. The useof methyl-labeled methionine provides a meansof differentially labeling the majority species ofRNA and other types of cellular RNA (13).

RNA-DNA HybridizationAt the present time, the only reasonable ap-

proach to the investigation of sequences of largeRNA molecules, which are indistinguishable bythe above-mentioned chemical tests, is to com-pare their ability to hybridize with DNA (41, 93).Especially useful is the so-called "competition"experiment, in which one species of RNA isshown to occupy or fail to occupy the same DNAsites as another species. As will be discussed later,in order to achieve clear-cut results in the com-parison of similar species of animal cell RNA byhybridization, care must be taken to measurerelatedness of molecules by the most exactingtests possible.

Cell FractionationAn important aspect in the study ofRNA mole-

cules is the determination of their cellular locali-zation. A number of significant advances in cellfractionation techniques have contributed to atleast a partial realization of this goal. Not onlyhave these techniques proved very useful inhelping to prepare various RNA species of greaterpurity, but strong evidence also exists that inmany cases they provide physiologically validfractionation of the cell (6, 67, 70). In manyexperiments, therefore, cell fractionation pre-cedes the release and study of RNA molecules.

WHOLE CELLTotal Cell RNA

One final point in this regard is that all cellu-lar RNA, with the possible exception of tRNA,exists in combination with protein within thecell. In the examination of cell fractions, it isfrequently advantageous to examine the typesand distribution of ribonucleoproteins of aparticular cell fraction before releasing theRNA for further physical or chemical analysis.The various experimental tactics and maneuvers

which will be referred to in the discussion tofollow are outlined in Fig. 1.

PATTERNS OF SYNTHESIS OF ANIMAL CELL RNA

"Rapidly Labeled RNA" and Kinetic AnalysisExperiments

About 80 of the RNA obtained from anytype of cell is made up of the two species ofrRNA. Most of the remainder is tRNA. In thesimplest of cases, exposure of growing cells toradioactive label would result in these species ofRNA becoming labeled in proportion to theiramounts within the cell. In fact, if either bacterialcells or growing animal cells are exposed tolabeled RNA precursors for a large fraction oftheir respective generation times, the majorityspecies of RNA do become proportionatelylabeled. Figure 2 shows that such a result occurswhen HeLa cells are labeled for 24 hr with 14C-uridine. If, however, the labeling period is re-stricted to a small fraction of the generation time(Fig. 3A), it is found that most of the recentlyincorporated radioactivity (i.e., newly madeRNA) is not in the majority species of RNA (85).Figure 3 shows the sedimentation pattern of thetotal HeLa cell RNA extracted from whole cellsafter labeling periods of 5, 15, 30, 45, and 60 min.The majority species ofRNA (28S, 16 to 1 8S, and

NUCLEUSNucleolus Nucleolor RNANucleoplasm Nucleoplasmic

RNATotalNuclearRNA

MITOCHONDRIONI Mitochondrial Associated RNA

-CYTOPLASMRibosomes aPolyribosomes

} Ribosomal andPolyribosomeAssociated RNA

FIG. 1. Diagram of cellular fractions or structures from which RNA call be extracted

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4S or tRNA) are seen to comprise the majorityof the UV-absorbing material. The labeled RNAclearly does not conform to the sedimentationprofile of the majority species. Several pointsare evident. (i) The rapidly labeled RNA isheterogeneous in sedimentation profile withmolecules sedimenting from about 20S all theway to 100S. (ii) In the sample taken from cellslabeled for 5 min, there appears to be a peak atabout 45S which becomes quite distinct after 15min of labeling. (iii) Between 15 and 30 min,radioactivity appears in a second prominent peakwhich sediments at 32S, somewhat faster than the28S or larger rRNA molecule. It is important topoint out here a fact which will be dealt with inmore detail later. Early experiments on radio-autography of animal cells which were brieflylabeled with radioactive RNA precursors hadestablished that the nucleus was the initial site ofincorporation of the great majority of 3H and14C purine and pyrimidine nucleosides (36, 73,77, 97). It was also established in early cell frac-tionation studies with L cells and HeLa cellsthat most (about 90%) of the radioactivityincorporated within 30 min of exposure tonucleosides was in the cell nucleus (20, 71). Thus,when the total "rapidly labeled RNA" was ex-tracted from whole cells, as in Fig. 3, it was clearthat the majority came from the cell nucleus.

Several possible origins of such "rapidly

A

800

400

FRACTION NUMBER

FiG. 2. Sucrose gradient sedimentation analysis oftotal cell RNA (HeLa cells) after a 24-hr exposure to14C-uridine. Fraction I is the bottom of the gradient inthis and subsequent diagrams; solid line, opticaldensity at 260 nm; 0, counts per minute. Redrawnfrom Scherrer and Darnell (85).

FIG. 3. Sedimentation analysis of radioactivity in total cell RNA (HeLa cells) after 5, 15, 30, 45, and 60 min(a-e) exposure to 3H-uridine. From Warner et al. (107).

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labeled nuclear RNA" could be immediatelysuggested. First, the RNA could be "nascent,"i.e., not yet completed polynucleotide chains.This possibility was obviously unlikely, at leastfor the great majority of "rapidly labeled RNA"from animal cells, because the rapidly labeledRNA had physical properties which indicatedthat it was substantially larger (had a greaterchain length) than the majority species of RNA.For example, samples of RNA isolated from

either the 45S or 70S region of a sucrose gradientagain sediment in the same region during a secondsedimentation (107). Treatment with deoxyribo-nuclease, proteolytic enzymes (trypsin and Pro-nase), ethylenediaminetetraacetate (chelatingagent), dimethyl sulfoxide (DMSO; destroyssecondary structure of macromolecules), orelevated temperatures failed to alter the sedi-mentation properties of the rapidly sedimentingmolecules (1, 52, 90,107). Electron micrographs ofmolecules taken from both the 45S and 60Sregions of the sucrose gradient provide a final bitof evidence on the chain length of the nuclearmolecules (88). The 45S rRNA is about two tothree times as long as the 28S rRNA, and the 60Smolecules are much longer yet (up to 5 to 6 ,um inlength), indicating molecular weights in excessof 107. Thus, it can be safely concluded that therapidly labeled RNA molecules are not aggre-gates or artifacts of any kind, but rather are verylong polynucleotide chains.A second explanation of the origin of the

rapidly labeled RNA could be that the solublepools of ribonucleotides from which polynucleo-tides are built were segregated. In this case, therapidly labeled material would represent a smallamount of the total cell RNA which was madefrom a pool that became labeled very rapidly incomparison with the pool from which the bulk ofthe cell RNA might be drawn. This explanationremains virtually impossible to test directly,but, as will become clear from the remainder ofthe discussion, it is most unlikely to prove accu-rate.

Strong experimental support has accumulatedfor a third explanation of the nature of "rapidlylabeled" RNA-namely, that these RNA mole-cules in animal cells represent a mixture of (i)larger molecules which are precursors to ribo-somal RNA and (ii) a class of RNA, about 1%of the total cell RNA, which is constantly beingsynthesized and degraded.

Before further describing experimental results,the importance of a "kinetic analysis" of the flowof radioactivity through and into various RNAspecies needs emphasis. Precursor-product rela-tionships of molecules within cells are difficult, ifnot impossible, to establish on kinetic grounds

alone (78). This is especially true of RNA mole-cules, because the introduction of radioactiveRNA precursors into the cell pool cannot befollowed by an effective chase (90, 107). [Ex-perimentally, this means that incorporation ofradioactive RNA precursor cannot be immedi-ately stopped upon addition of unlabeled pre-cursor to the medium. This is in fact true forbacterial as well as animal cells (63).] Thus,without the intervention of drug treatments, it isnot possible to label precursor molecules, stopfurther labeling, and observe the fate of thelabeled species. To discover something about theprecursor relationship of rapidly labeled speciesto the major RNA species, another approach hasbeen necessary. First, chemical evidence was ob-tained of a relationship between rapidly labeledRNA species and a majority species of RNA.Second, it was determined how long it takes toaccumulate the maximal label in a rapidly labeledspecies before the first appearance of label invarious chemically related molecules.Chemical evidence about the rapidly labeled

RNA molecules was most easily obtained bylabeling cells with 32PO43-, isolating variousRNA species by zonal sedimentation, and de-termining the base composition of RNA mole-cules with different sedimentation coefficients.Figure 4 shows such an experiment. The totalcellular RNA of HeLa cells labeled with 32PO43-for 35 min was examined. The most importantpoints which come from this experiment are thefollowing. (i) The sedimentation profile oflabeled RNA molecules is approximately thesame as a 20-min uridine label (compare with Fig.3) due to the slower entry of P043- into the acid-soluble nucleotide pool. (ii) The guanine pluscytosine (GC) content of the molecules from the45S region is high (>60%). (iii) The largermolecules (those sedimenting from 45 to -90S)have a low GC content. Comparison of thesevalues with the majority species of RNA, rRNAand tRNA, and with HeLa cell DNA (Table 1)reveals that the heterogeneous large RNA has acomposition similar to DNA. For convenience,this material has been designated as heterogene-ous nuclear RNA, HnRNA (90, 107). (Otherauthors have referred to this RNA as "giantRNA," "messenger-like" RNA, or dRNA; also,see references la, 24, 49, 87, 112).By contrast, the RNA in the 45S region was

found to have a base composition similar to therRNA (86, 90, 107). These results, of course,suggested a possible precursor role for the 45SRNA in the formation of ribosomes. A decisiveexperiment to test this hypothesis was madepossible by the discovery of the action of actino-

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FIG. 4. Sedimentation and base composition anialysis of total "rapidly labeled" HeLa cell RNA after 35-mimlexposure to 32P-orthophosphate. Samples were taken of various sizes ofRNA after the sedimentationi analysis anidexamined for base composition after alkaline hydrolysis. Results are plotted in the bar graphs as percentage guanylicand cytidylic acid (%GC). From Soeiro, Birntboim, antd Darnell (90).

TABLE 1. Distributioni and base compositioni ofvarious molecules in HeLa cellsa

Cellifraction

Cyto-plasma

Total

Nucleus

RNA species

28S rRNAl7S rRNA5S rRNA18S rRNAtRNA"pre-tRNA"mRNA (poly-somes)Cytoplasmicheterogene-ous,

45S r-pre-RNA32S r-pre-RNAHnRNA

_g Base composition,_< % as

0S C A G U GH

53 32 16 36 16 6,- 28 21 28 235-1 26 18 34 22624 27 21 30 22 c12 27 22 27 24 c<1

1-l3<1

94.5

1

3

24 26 211 28

333322

131426

373721

171631

a Data taken from references 52, 90, and 107.The base composition of HeLa cell DNA is C,21; A, 29; G, 22; T, 28. Abbreviations: pre-tRNA,precursor transfer RNA; r-pre-RNA, ribosomalprecursor RNA; HnRNA, heterogeneous nuclearRNA; C, A, G, U, and T represent cytidylic,adenylic, guanylic, uridylic, and thymidylic acids.

bHeterogeneous RNA not in polysomes.

mycin D, an antibiotic which binds to DNA,thereby preventing RNA transcription by RNA

polymerase (50, 79). Thus, cells which had beenlabeled long enough so that the 45S peak wasvery prominent but not long enough for radio-activity to be found in 28 and 18S rRNA weretreated with actinomycin to determine the fateof 45S RNA in the absence of further RNAsynthesis. It was found (Fig. 5) that the 455RNA quickly disappeared in actinomycin-treated cells, and coincident with its disappear-ance radioactivity appeared in 32 and 185 RNA(35, 67, 86). After longer periods in actinomycin,the radioactive RNA in the 32 to 28S regiongradually shifted to predominantly 28S (35).These two experiments a determination of

the base composition of 45S RNA and the"actinomycin chase"-strongly suggested thatrRNA in HeLa cells was formed as a long poly-nucleotide which was subsequently cleavedspecifically to yield the 28 and 18S rRNA mole-cules. Thus, two classes of molecules make upthe majority of the rapidly labeled nuclear RNA-the HnRNA, which is DNA-like in composi-tion, and 45S RNA, which is related to rRNA.There are also other types of cytoplasmic RNAwhich make up only a small proportion of thetotal rapidly labeled RNA; these will be discussedlater.

BIOSYNTHESIS OF RIBOSOMES IN ANIMAL CELLS

Ribosomal Precursor RNASince a great deal of morphological and cyto-

genetic evidence suggested that the nucleolus

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FIG. 5. Actinomycin chase experiment. HeLa cells were exposed to 3H-uridine for 25 miii (A), 45 miti (B), or 60min (C). At 25 min, the culture was divided and a portion was treated with actinomycin (5 Ag/ml). Samples of thetreated culture were taken after 10 min (D), 20 min (E), or 35 min (F). Total cell RNA was extractedfrom eachsample and analyzed on sucrose gradients.

was the site of nuclear ribosome accumulation ifnot also of formation (4, 8, 14, 22, 74), attemptswere made in cultured cells to prove that 45SrRNA existed in the nucleolus. The first stronglysuggestive experiments on this point were per-formed in L cells using a combination of radio-autography, actinomycin D treatment, andexamination of extracted RNA from L cells (72).First, it was shown that growing L cells synthe-sized 45S RNA and HnRNA and that radio-autography revealed strong incorporation inboth the nucleolus and the nucleus at large. Brieftreatment of these cells with a low dose of actino-mycin (0.05 ,ug/ml) reduced RNA precursor up-take by about 50%. However, nucleolar RNAsynthesis, as observed by radioautography, and45S ribosomal precursor RNA (r-pre-RNA)synthesis, assayed by zonal sedimentation, werealmost completely obliterated. It was thereforeconcluded that 45S synthesis occurred in thenucleolus of the cell. Cell fractionation studieshave added additional evidence that this is thecase. HeLa cell nuclei can be lysed by deoxyribo-nuclease treatment in the presence of relativelyhigh salt concentrations (0.5 M NaCl, 0.05 MMg+), leaving the nucleoli intact morphologic-

ally so that they can be easily isolated (47, 70).Release and examination of the nucleolar RNArevealed that about 2 to 3% of the total cell RNAcould be recovered as 45S and 32S r-pre-RNA(47, 70; R. Soeiro et al., J. Cell Biol., in press).(See Fig. 12 and Table 1.) Moreover, no 45S or32S r-pre-RNA was found outside the nucleolus(Fig. 6; 47, 70, 90). Thus, a perfect correlationexisted between the cytological and cytogeneticevidence and the biochemical evidence thatribosome formation in animal cells was initiatedby the formation of 45S r-pre-RNA in the cellnucleolus.Another important chemical event in the manu-

facture of ribosomes has been shown to occur inthe nucleolus. The rRNA isolated from HeLacells (total cells) had been found to containmethyl groups both attached to nucleic acid basesand to the 2' OH of the ribose of rRNA (13).The direct precursor of RNA methylation reac-tion was known to be the terminal methyl groupof S-adenosyl methionine. Using methyl-labeledmethionine as a precursor for these methyl groups,it was shown (Fig. 7) that the first molecules inthe pathway to ribosomes which become labeledwere the 45S r-pre-RNA followed by 32S (later

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FRACTION NUMBER

FIG. 6. Partition of radioactive RNA in fractions of HeLa cells. Cells were exposed to 14C-uridine for 10, 30,50, or 70 min andfractionated; RNA was extracted and analyzed. From Penman, Smith, and Holtzman (70).

28) and 18S, just as was the case when nucleo-sides were the labeled RNA precursor (37, 115).Thus, the 45S ribosomal precursorRNA appearedto be the chief, if not the sole, site of methyla-tion. For example, if 50% of the methylationoccurred on rRNA molecules past the 45Sstage, then methyl incorporation should haveproceeded in those species as well as in 45S. Noevidence of methyl incorporation directly into32, 28, or 18S regions was found. In addition,methylation apparently occurred either verysoon after or concomitant with 45S synthesis,because actinomycin D treatment abolishedwithin 5 min the ability of the cells to incorporate

methyl groups into 45S RNA (115) or any otherhigh molecular weight RNA.Thus far, experiments have been described

which indicate that 45S ribosomal precursorRNA is made and methylated in the nucleolus.Since 32S RNA is also found in the nucleolus, itappears that the cleavage of the 45S r-pre-RNAalso begins in the nucleolus. Recent experiments,in fact, indicate that a whole series of cleavagesteps occur in the nucleolus. These experimentswere made possible by the development of thetechnique of acrylamide gel electrophoresis ofRNA molecules (59), which separates single-stranded RNA molecules, presumably on the

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-j II.3 - 3 3.0

w I

10 20 30 10 20 30 10 20 30FRACTION NUMBER FRACTION NUMBER FRACTION NUMBER

FIG. 7. Incorporation of methyl groups inito 45S ribosomal precursor RNA. HeLa cells were labeled for 10min with '4C-methyl labeled methionine and RNA was extracted from cell fractions. From Greenberg and Pen-man (37).

basis of chain length, but which affords some-what higher resolution of RNA species than doessucrose gradient zonal sedimentation. Whentotal nucleolar RNA was extracted from cellswhich had incorporated labeled methionine for 30min, it was found by gel electrophoresis analysisthat, as expected, the 45S peak was the majorlabeled species (109). Moreover, the optical den-sity tracing of the gel showed peaks between 45and 32S, which indicated the possibility of a dis-tinct species intermediate between the two mainrRNA precursors (Fig. 8). (For convenience,these intermediate peaks are designated 41S and36S, although no careful sedimentation analysis ofthem has been performed.) When cells labeled for16 min with methyl-labeled methionine were thenexposed to actinomycin, and nucleolar RNAsamples were examined 9 and 20 min later, radio-activity was seen to shift through peaks at 41and 36S before appearing at 32S (Fig. 9). Inaddition, a 205 molecule was labeled in advanceof the 185 molecule. One further observationcame from this series of experiments: definiteevidence was obtained that the earliest labeled28S RNA was located in the nucleolus.Another set of experiments has demonstrated

one additional nucleolar event in the processingot ribosomal precursor RNA. It was found that28S rRNA, regardless of the method of isolation,was not one covalently linked polynucleotidechain. Upon exposure to heat, urea, or DMSO(all procedures which disrupt hydrogen bonding),the "28S" RNA gave rise to a small moleculeabout 150 nucleotides in length which has beentermed 7S rRNA. It was further found that in

0.D260 CPM r--1.5 700r--

600!--

500L~1.0k

400--

0.5k

2001--

00t---

,45S

10 20 30SLICE NUMBER

FIG. 8. Acrylamide gel analysis of nucleolar RNA.Nucleoli from HeLa cells which had been labeled with'4C-methyl methionine for 30 miii were prepared, andRNA was extracted and subjected to separation byacrylamide gel electrophoresis. Solid line, opticaldensity at 260 nm; 0, counts per minute. From Wein-berg et al. (109).

nucleolus orRNA after urea treatment an amountof this 7S RNA existed which was equivalent innumber of molecules to the amount of nucleolar28S RNA (66a).The present scheme of the nucleolar cleavage

of rRNA is summarized in Fig. 10. The exact

CPMH3

. 2500

2000

1500

1 1000

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40 50


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c14F-14~~~~4nCPM 16min. C Meth +9min. Actino +26 min. Actino

100---45SI

80 32S

60'-

40

vo0-

32S45S

10 20 30 40 10 20 30 40

SLICE NUMBER

FIG. 9. "Actinomycin chase" of methyl-labeled RNA. The nucleolar RNA of HeLa cells labeled with 14C-methyl methionine and subsequently chased with actinomycin was analyzed by acrylamide gel electrophoresis.From Weinberg et al. (109).

45S 32S 28:7(4.5 x 106)l 41 36 1(2.4 x 06) (1.9 x j6)

~~~~7S('

Degrac(0l(0.5x

Degraded(1.4x 106)

l ~~IB,____- i0718S

(0.7x 106)

4x 104)

ded106 )

FIG. 10. Summary of the processing of 45S r-pre-RNA. This diagram indicates the molecular weightsand approximate S values of various ribosomal-relatedRNA moleculesas well as thefate ofsomeportions of theRNA which do not survive processing.

point during the processing at which the 18Smolecule is released is not clear, but it is presentby the time the 32S is observed.

These considerations of 45S r-pre-RNA cleav-age raise a number of important questions. Forexample, what is the molecular weight of 45Sr-pre-RNA, and does it contain one 28S and one

18S molecule? Is the entire 45S molecule used inconstruction of ribosomes? What determines thespecificity of the cleavage points in the poly-nucleotide? Answers to a number of these ques-tions are now at hand.

Since different RNA molecules form randomcoils of varying degrees of compactness at a

given salt concentration (95, 107; J. H. Strauss,Ph.D. Thesis, California Institute of Technology,Pasadena, 1966), it is not possible to obtainreliable molecular weights of RNA species byzonal sedimentation alone. The technique ofsedimentation equilibrium determination ofmolecular weight, however, circumvents thisproblem, because molecular shape is relativelyunimportant in such a determination (113). Theavailability of nucleolar preparations from which45S and 32S r-pre-RNA could be obtained pure inrelatively large amounts has allowed a determina-tion of their molecular weights by sedimentationequilibrium. The molecular weight of the mainrRNA species, 28 and 18S, has also been meas-

ured in the same manner, and the data are re-

ported in Fig. 10 (E. D. MacConkey and J.Hopkins, J. Mol. Biol., in press). It is clear thatonly one 32S (2.4 X 106) and one 18S (0.7 x

28S

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106) molecule could be drawn from each 45S(4.4 x 106) precursor molecule. After subtract-ing the 32S and 18S from the 45S, there is someRNA (approximately 106 daltons) which is leftover; likewise, in the 32 -+ 28S conversion asubstantial amount (-5 X 105 daltons) of RNAis not included in the final rRNA product.Several types of chemical evidence agree withthese physical measurements which indicate thatonly 50 to 60% of the 45S is actually conservedduring processing (1, 52, 99, 101). (i) A com-parison of the oligonucleotide maps obtainedafter partial enzymatic digestion of 45S r-pre-RNA, 32S r-pre-RNA, 28S rRNA, and 18SrRNA reveals that the precursor moleculescontain a qualitatively and quantitatively differ-ent makeup than the 28S plus 18S rRNA inabout 50% of their length (1, 52). (ii) Aspreviously mentioned, it was shown that the onlyacceptor of methyl groups was 45S r-pre-RNA,and in addition all the incorporated methylgroups in 45S r-pre-RNA were conserved duringfurther RNA processing (101). Thus, with RNAsamples labeled both with "4C-methyl groups andwith 3H-uridine (which would label pyrimidinebases throughout the chain), a test could be madeof whether nonmethylated portions of the RNAchains were lost (109). If unmethylated RNA wasdegraded to acid-soluble material as 45S was proc-essed, but all methyl groups were conserved, thenthe "4C-methyl/3H-pyrimidine ratio would in-crease during processing. Table 2 shows that thiswas indeed the case, indicating a loss of abouthalf of the 45S RNA during processing. (iii) Athird type of evidence, very similar to the second,was obtained by assaying various RNA specieslabeled with nucleosides for the content ofalkali-resistant dinucleotides. This provides a

TABLE 2. Extent of methylation of r-pre-RNAcompared to rRNA

RNA species Uridine/methyl ratioa Dinucleotidesb (%)

45S 1.59 0.8632S 1.16 1.1028S 0.79 2.0818S 0.55 2.74

a RNA of various sizes from cells labeled withboth l4C-uridine and 3H-methyl methionine showsthe increasing methyl content of rRNA comparedto precursor RNA (109).

bRadioactive RNA of various sizes labeled inuridine and cytidine residues was hydrolyzed byalkali. A measurement of the relative amounts ofdinucleotides indicates the frequency of methyla-tion of 2' OH groups on ribose. From Vaughanet al. (99).

measure of the relative content of methylation ofthe 2' OH groups of ribose in various RNAspecies. The dinucleotide content was found tobe 45S < 32S < 28 and 18S. Since -CH3groups are inserted only into 45S RNA, theseresults also indicate a loss of material lackingmethylated ribose as RNA maturation pro-ceeded (99).These experiments indicate then that about

50% of the 45S r-pre-RNA is made up of nucleo-tide sequences that are used to make ribosomes,and the other half of the molecule is synthesizedand apparently then destroyed because there isno accumulation of any RNA except the 28 and18S resulting from the processing of the 45Smolecule.Another question which was repeatedly raised

about the 45S r-pre-RNA was whether only asingle class of precursor molecules exist contain-ing both the 28 and 18S. Chemical evidencecertainly implies that both 28 and 18S are drawnfrom 45S molecules: (i) 45S r-pre-RNA, the solesource of high GC RNA in briefly labeled cells,is destroyed in actinomycin with the resultingdevelopment of 28 and 18S RNA (35, 72, 86,107); (ii) oligonucleotides of both 28 and 18Stype are found in 45S molecules (1, 52); and (iii)the characteristic distribution of methylated basesfor both 28 and 18S RNA is represented in 45SRNA (101).A recent experiment performed with nucleic

acids from amphibian tissues strongly indicatesthat the DNA molecule from which 28S RNAarises also carries the information for 18S RNA.This experiment was performed by isolatinghybrids between frog DNA and 28S RNA anddemonstrating that the DNA so isolated also hasan increased capacity to hybridize 18S RNA (12).

It can be concluded that the 45S r-pre-RNAis a large polynucleotide of about 4 x 106 daltonswhich contains one 28S and one 16S molecule inaddition to some unmethylated stretches ofunknown function.

Formation and Distribution ofRibosomal Particles

Thus far, our discussion has been concernedonly with experiments in which rRNA has beenstudied after being freed from combination withprotein. Within the cell, rRNA is always foundin combination with protein, and we will nowmove to a discussion of the formation and dis-tribution of ribonucleoprotein particles incultured cells. Cellular homogenization, eitherin isotonic sucrose or in hypotonic solutions, hasbeen employed for a number of years to release alarge fraction of the cytoplasm of animal cells

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(liver cells and thymus cells in particular) withoutdisrupting nuclei (6, 21, 45). When HeLa cellsare exposed to hypotonic homogenization andthe resulting cytoplasmic extract is examined byzonal sedimentation for its content of RNA-containing (UV-absorbing) particles, a numberof structures are observed (Fig. 11; 34, 69). Thewell-known sedimentation behavior of singleribosomes (75 to 80S) can be used as a guide indescribing the other structures (98). The majority(about 75 to 90%) of the UV-absorbing materialsediments faster (-1OOS to 400S) than thesingle ribosome. These structures have beenidentified in a variety of ways as polyribosomes,groups of ribosomes which are attached to thesame mRNA molecule actively synthesizingprotein (31, 64, 104). Approximately 10% of theribosomes in the extracts of exponentially grow-ing HeLa cells sediment as free single ribosomeswhich at the time the cell is broken are notengaged in protein synthesis (34). In addition tothese classes of "whole ribosomes," there are alsoobserved in the optical density tracing two peakswhich sediment more slowly than whole ribo-somes. By extracting the RNA from thesestructures, it was concluded that these were freesubunits-a 60S ribosomal subunit, containing28S rRNA, and a 40 to 45S ribosomal subunit,containing 18S rRNA (34, 53, 98).

After the distribution of cytoplasmic ribosomeswas established, experiments were designed todetermine in which form newly made ribosomalparticles would appear in the cytoplasm. Cellswere labeled, fractionated, and examined for the

distribution of radioactive RNA in variousfractions. It was found that the first appearanceof both 18S and 28S RNA was in the form offree subunits (Fig. 12; 34, 53). Within 10 to 15min of the appearance of new subunits, ribosomescontaining newly formed RNA could be foundin polyribosomes without radioactivity appearingas a peak in the single ribosome. Thus, it ap-peared possible that the new ribosomal subunitsentered into protein synthesis directly, that is,without first becoming single ribosomes. Theobligatory initiation of protein synthesis by

QD

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FIG. 11. Sedimentation of cytoplasmic extracts ofHeLa cells. Extracts were prepared by homogeniza-tion of cells swollen in hypotonic buffer (69), and threeconditions for sedimentation analysis are demonstrated.Left, extract was sedimented for 16 hr at 20,000 revlmin, 4 C, through a 28-ml sucrose gradient (15 to30%, wlw). Center, extract was sedimented for 90min at 25,000 rev/min 4 C, throughi a 28-ml sucrosegradient (15 to 30%, wlw). Right, extract was sedi-mented for 5 hr at 25,000 rev/mim, 4 C, through a30-ml sucrose gradient (7 to 47%, w/w).

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FiG. 12. Appearance of new ribosomal subunits in HeLa cell cytoplasm. Cytoplasmic extracts of cells labeledwith 3H-uridine for 30 (A) or 60 (B) min were sedimented through a sucrose gradient (conditions similar to Fig. 11left panel) anzd the gradient contents were assayed for acid-precipitable radioactivity. Inset in part A shows thatRNA removedfrom labeled peak of ribonucleoprotein at 45S is mostly 16S rRNA. From Girard et al. (34).

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subunits has recently been demonstrated inbacteria (65, 89).The new subunits in mammalian cells have

been shown to be distinguishable from thepre-existing free subunits because they have ahigh protein/RNA content and thus band at aless dense region in CsCl (75). By the time theyenter polyribosomes, however, they have thesame density as the other ribosomes participatingin protein synthesis. The meaning of this changein relative protein content is not known. Even-tually, all the cytoplasmic ribosomes of thecell-polysomes, single ribosomes, and subunits-come into equilibrium. This was shown byexperiments in which cells were labeled and thengrown for a long time in unlabeled medium; thespecific activity of the ribosomal RNA in allparticles was then the same (100).One very suggestive result from these earlier

experiments on ribosomal particles in culturedcells was the finding that new ribosomes enteredthe cytoplasm as subunits, not as whole ribo-somes. The possibility was raised that nothingbut subunits existed within the nucleus.The simple hypotonic swelling and homogeni-

zation technique that had been employed in theearlier experiments with HeLa cells gave variableresults in the recovery of rRNA (50 to 90% inthe cytoplasmic fraction; 69). Therefore, atechnique was sought which would break all thecells, leave the nucleus intact, and leave thesmallest amount of contaminating cytoplasmwith the nuclear preparation. This latter con-sideration is of great importance, since manyworkers had pointed to the large number ofribosomes near or attached to the nuclear mem-brane (4, 6). Penman, Smith, and Holtzmansucceeded in developing a technique for HeLacells which employed hypotonic swelling andhomogenization for the removal of the majorityof the cytoplasm, followed by a combinedionic-nonionic detergent treatment of the nuclei.This latter step removed the nuclear rim ofcytoplasm and the outer layer of the nuclearmembrane without altering the intranucleararchitecture (Fig. 13). When nuclei which hadbeen "detergent cleaned" in this manner wereisolated from cells which had first been labeledwith pyrimidine nucleosides and then grown forseveral generations in unlabeled medium, theywere found to contain almost none (less than5%) of the total cell 18S rRNA and by inferenceless than 5% of the cellular ribosomes (67). Theystill contained all of the nucleolar RNA, however,as well as the nuclear DNA. Moreover, thenucleoli looked intact and still had a granular

appearance which had been attributed to "nu-clear ribosomes" (47, 67, 70).A search was therefore made in detergent-

cleaned nuclei for ribonucleoprotein particlescontaining rRNA, with the knowledge that therewould probably be relatively few such particles.In addition, it was anticipated that any nuclearribosomes might represent newly synthesizedparticles which would therefore be the firstribosomal particles labeled in the cell. In experi-ments on the preparation of nucleoli referred toearlier, it was pointed out that the clean nucleican be lysed by exposure to high ionic strength(0.5 M NaCl + 0.05 M MgCl2) in the presence ofdeoxyribonuclease. After removal of the nucleoliby brief centrifugation, these nuclear extractswere examined for ribosomal particles in com-parison with cytoplasmic extracts treated in thesame manner (100). A reproducible number ofparticles with the sedimentation characteristicsof larger ribosomal subunits (50S in high ionicstrength) were observed in nuclear extracts.These particles were then shown to contain 28SRNA. A final proof that they were truly nuclearparticles was obtained by fractionating cellsafter various periods of labeling to demonstratethat radioactive 28S rRNA appeared in thesenuclear particles before any could be detected inthe cytoplasm (Table 3). A quantitation of theamount of 28S rRNA in these nuclear precursorparticles indicated that they contained about 1 %l,as much 28S rRNA as was present in the totalcytoplasm. Evidence was also obtained that 18SRNA could be isolated from nuclear subribo-somal particles before it was observable in thecytoplasm. However, the speed of exit to thecytoplasm of particles containing 18S rRNA wasmuch greater than with the 28S-containingparticles.These experiments established the existence of

an intranuclear stage of ribosome developmentwhen the ribosome is probably largely complete-i.e., has approximately the same sedimentationbehavior as a mature subunit. The questionnaturally arises of whether these nuclear particlesare complete with respect to proteins and whetherit is possible to isolate other particles which areless completely formed. The topic of ribosomalproteins is too large to be dealt with extensivelyhere, but a few points from this area of researchare necessary to further consideration of theassembly of ribosomal structures.

Ribosomal Proteins and Nucleolar RibosomalPrecursors

Ribosomal protein and ribosomal RNA arenot held together by covalent bonds. Thus, it is

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possible, depending on a variety of conditions(pH, ionic strength, Mg++ concentration, etc.),to isolate rRNA in combination with variableamounts of protein (66). The first decision in

dealing with ribosomal protein, therefore, is todecide what belongs with rRNA, or at least toadopt techniques which allow reproducibleisolation of ribosomes containing a reproducible

FIG. 13. Electroii micrographs ofHeLa cell ,iucleoli. All preparations were fixed in glutaraldehyde and stainedwith uranyl acetate and lead citrate. (a) Untreated HeLa cell. The nucleus occupies most of the field. N indicatesa nucleolus. X 8,000. (b) Cell swollen in hiypotonic buffer. The nucleus occupies most of the field. Its contentsappear considerably more homogeneous thanz in untreated cells. The ntucleoli are less prominent except for theintranucleolar strands which stand out sharply (arrows). X 7,000. (c) Nucleus isolated after hypotonic swelling,homogenization, and detergen2t treatment. The outer nuclear envelope is no longer seen at the surjtice (arrow).Nucleoli are present. X 7,000. (d) N indicates a nucleolus isolated from a preparation as in part c. Thle overallform of the nucleolus has been preserved through the isolation, and many small granules, possibly ribonucleoprotein,are present withint. X 20,000. Photographs courtesy Eric Holtzmal, similar to those published in Holtzman, Smith,and Penman (47).

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TABLE 3. Content of labeled 28S rRNA in nuclearand cytoplasmic particlesa

Radioactivity (counts/min) in

Label time 28S rRNA ratio,Label(imi) nucleus/(min)cyolsNuclear 50S Cytoplasmic 60S cytoplasm

particles particles

37 >500 Not detected42 >1,500 <150 >1090 8,000 8,600 0.91

Steady state 0.1-0.15

a After various exposure times to ('IC) uridine'the amount of radioactive 28S rRNA in nuclearand in cytoplasmic subribosomal particles wasdetermined (99).

set of proteins. Warner has studied the condi-tions necessary for producing HeLa cell ribosomesuncontaminated by large amounts of extraneousprotein (105). In addition, he has characterizedthe ribosomal proteins by gel electrophoresis sothat a number of different characteristic proteinpeaks can be identified (102). Two functionalclasses of HeLa cell proteins are of interest forour discussion. (i) Since ribosomal RNA origi-nates in the nucleolus and after some delayarrives in the cytoplasm in the form of a ribosomalsubunit, experiments were designed to determinewhether newly formed ribosomal protein alsounderwent a delay between synthesis and ap-pearance. It was found that a large fraction ofthe ribosomal protein did in fact behave this way,i.e., appeared in cytoplasmic ribosomes onlyafter a 30- to 120-min delay. This fraction will bereferred to as structural ribosomal protein. (ii) Asecond class of proteins, about 20% of the totalribosomal protein, is regularly found on HeLacell ribosomes but shows little or no delaybetween synthesis and appearance in ribosomes.Moreover, the synthesis and association withcytoplasmic ribosomes of this second class ofribosomal proteins continues even after cells aretreated with actinomycin, a treatment whichstops the synthesis of rRNA immediately andthe appearance of new cytoplasmic ribosomeswithin 1 hr (35). This second class of ribosomalproteins will be termed cytoplasmic ribosomalprotein to indicate that they exchange withribosomal particles in the cytoplasm.One further important point should be made

about the ribosomal protein of HeLa cells. Asstated, the structural ribosomal protein does notappear in the cytoplasm for about 30 to 120 minafter it is synthesized. This indicates that a poolof structural ribosomal protein exists throughwhich new material must pass before beingobserved on cytoplasmic ribosomes. An addi-

tional experiment which indicates that such aprotein pool exists has been reported. If cells aretreated with cycloheximide, a drug which com-pletely blocks all new protein synthesis, they arestill able to synthesize new rRNA which becomesassociated with ribosomal protein to formapparently functional ribosomes (103). Also, atleast 80% of the labeled protein which appearsin ribosomes after a pulse and cold amino chasewill also appear in cytoplasmic ribosomes in thepresence of cyclohexamide. Thus, the ribosomalprotein pool can be used by rRNA synthesized inabsence of ongoing protein synthesis.

Association of Ribosomal Protein with rRNAWe can now address the question of when, in

the course of ribosome formation, rRNA andribosomal protein come together. We previouslypointed out that nuclear particles with sedi-mentation properties similar to cytoplasmicsubunits were identified in nuclear extracts afterremoval of the nucleoli. It is not known whetherthese particles were part of the nucleolus insidethe cell and were dislodged by the high ionicstrength deoxyribonuclease treatment. For ex-ample, particles of the appropriate size for thelarger ribosomal subunit have been seen re-peatedly by electron microscopy around theborder of the nucleolus (4, 76), and these mighteasily be lost in purification of the nucleus. Atany rate, HeLa cell nucleoli prepared by thehigh ionic strength deoxyribonuclease methodare relatively free from these mature particlescontaining 28S RNA. Recently, a technique hasbeen worked out for solubilizing the nucleolus,which results in the release of yet another class ofribonucleoprotein particles with sedimentationcoefficients of approximately 80S and 55S (Fig.14; 106). The RNA from these particles hasbeen shown to be ribosomal precursot RNA, notmature rRNA. In addition, gel electrophoreticanalysis of the protein of these nucleolar particlesshows that their protein corresponds in con-siderable detail to the structural ribosomalprotein of cytoplasmic ribosomes. The ribosomalproteins which are added in the cytoplasm arenot found in the nucleolar particles. Theseexperiments demonstrate that the association ofribosomal proteins and rRNA begin immediatelyafter, or even coincident with, the synthesis of theribosomal precursor RNA. A summary ofribonucleoprotein particle formation in theHeLa cell is given in Fig. 15.

Place of Small RNA Molecules in RibosomalMaturation

Within the past several years, a number oflaboratories have reported the existence of a

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-0.4 10,OOOH

0.2 5000

30

120

110

277

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I0x

I

FIG. 14. Nucleolar ribonucleoprotein particles. Upper panels: HeLa cells were labeled either with H4C-leucine(A) or '4C-uridine (B) and nucleoli were isolated and disrupted in buffer containing EDTA and dithiothreitol.Ribonucleoprotein particles were displayed by sucrose gradient analysis. The two peaks are approximately 80Sand 55S. Lower panels: RNA from 55S (A) and 80S (B) nucleolar particles was released and cosedimented withtotal nucleolar RNA. From Warner and Soeiro (106).

small rRNA, termed 5S RNA, one molecule ofwhich is found in every larger ribosomal subunit(19, 28, 81). It has been determined that in HeLacells this 5S RNA is contained within all themature ribosomal particles containing 28S rRNA(55). In addition, the 5S RNA has also beendetected in the newly described nucleolar particlescontaining ribosomal precursor RNA (106). Itis of interest, however, that the 5S RNA isapparently not part of the 45S r-pre-RNA. Theclearest evidence of a separate origin of r-pre-RNA and of 5S is that the DNA to which 5S

RNA hybridizes is separable from that withwhich rRNA hybridizes (12). In addition, it wasshown that the 28S molecule in a newly formedHeLa cell ribosome is not accompanied by a new5S RNA molecule. This result indicates a "pool"of 5S RNA through which new molecules mustpass before entering into ribosome manufacture(55).

Control of Ribosome Formation

The control of the rate at which new ribosomesare formed may be assumed to be important

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NUCLEUSNUCLEOLUSI NUCLEOPLASM

'45

I I<32 ~2:

NASCENT NEWRIBOSOMAL RIBOSOMALPARTICLES SUBUNITS

CYTOPLASM

or

~~~~~~~~~~~.28:7~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

NEW a OLDRIBOSOMALSUBUNITS

POLYSOMES SINGLERIBOSOME

FiG. 15. Summary of ribosome formation in HeLa cells. Solid shapes indicate ribonucleoprotein structures con-taining rRNA or ribosomal precursor RNA plus ribosomal protein. Points of detection of 5S and 7S RNA areindicated. Althouigh the ribosomal subunit containing 18S RNA is indicated as arising from the structure bearing36S RNA, this is not definite and may occur earlier.

because of the central role occupied by ribosomesin protein synthesis and, therefore, in cellularmetabolism. Two recent lines of experimentationhave made clear that various means are utilizedin animal cells to influence the rate of ribosomeformation. Let us first consider a case wherevastly accelerated ribosome biosynthesis occurs.In developing oocytes of frogs and salamanders(12, 29), it has been noted morphologically thatmultiple large nucleoli exist and that rapidincorporation of isotopes into ribosomal pre-cursor RNA and ultimately into ribosomes canbe demonstrated. Since one of the components ofthe nucleolus is probably the DNA from whichribosomal precursor RNA is formed and sincethe oocyte nucleoli are so prominent, attemptswere made to determine whether there existed inthese cells increased amounts of DNA com-plementary to rRNA. It has now been demon-strated in both types of oocytes that the amountof DNA complementary to rRNA has increasedperhaps as much as 100-fold compared to DNAfrom other tissues or whole animals. Thus, inthese selected cases, the need for rapid synthesisfrom ribosomal genes has been met by greatlyincreasing the number of genes (11, 12).

After oocytes reach the stage of mature eggs,they no longer can be demonstrated to be makingribosomes. Even after fertilization, no newribosome synthesis occurs. This apparent com-plete lack of ribosome formation is accompaniedby a lack of formation of 45S precursor RNA.These findings have been reported not only forfrog eggs but also for sea urchin eggs (11, 39).

These results appear to indicate a completedormancy of rRNA genes, the antithesis of thefirst control situation.

Perhaps a more common need encountered inanimal cells from various tissues is not a com-plete suppression of ribosome formation butrather a fluctuating rate of synthesis. For example,the cell life span in liver and in kidney is verylong, yet these cells incorporate isotopes intoribosomal RNA (43, 58, 61). Since the amountof tissue and ribosomal mass in adult liver andkidney is essentially constant, the likely explana-tion of such findings was that ribosomes werebeing constantly synthesized and degraded, i.e.,"turned over." The half-life of ribosomes inresting liver tissue, in fact, has been estimated at4 to 6 days (58). If a portion of the liver or akidney is removed from rats, the remainingtissue-liver or kidney undergoes very rapidgrowth, including a sustained synthesis andincrease in total ribosomal mass (17, 61). Whileit is clear in these situations that the rate ofaccumulation of new ribosomes is greatly en-hanced compared to resting tissue, it is notclear at what stage in the complicated chain ofevents leading to ribosome formation thisincrease has been effected. For example, it ispossible that the rate of 45S r-pre-RNA synthesisis unchanged but, in normal liver or kidney, it isnot all processed into ribosomes.The potential focal points of control of ribo-

some biosynthesis can possibly be studied moreeffectively in cultured cells where the stages ofribosome production can be more easily ob-

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served. Therefore, experiments were undertakento compare ribosome formation in rapidlygrowing cultured cells with cells which have hadtheir growth slowed or stopped in a variety ofways.

It was previously mentioned that cyclohexi-mide, a drug which essentially stops polypeptidechain elongation (26, 110), does not immediatelystop ribosome production by HeLa cells (103).These results allow two conclusions: (i) there is apool of ribosomal proteins which can be used toconstruct ribosomes in the absence of ongoingprotein synthesis; (ii) r-pre-RNA has no neces-sary role in directing protein synthesis in order tobe processed into a ribosome. All cells, however,are not equally capable of producing ribosomesafter cycloheximide treatment. For example,after cycloheximide treatment, L cells cancontinue to synthesize r-pre-RNA and process itinto 28S rRNA which appears in cytoplasmicribosomal particles, but the smaller or 18S rRNAis lost in the process (25).Treatment of HeLa or L cells with puromycin,

another drug which interrupts protein synthesis,has a drastically different effect from cyclohexi-mide on ribosome formation (56, 91). Althoughr-pre-RNA continues to be made after puromycintreatment, it cannot be effectively incorporatedinto a ribosome. Thus, a condition is inducedwhere synthesis and degradation proceed inparallel. The speed of degradation of the smaller18S rRNA is especially obvious, there being nosign of labeled 18S ever observable in either thenucleus or cytoplasm in cells labeled with 3H-uridine after puromycin treatment. There is noevidence of a defect in the r-pre-RNA synthe-sized in the presence of puromycin, since RNAwhich is made in the presence of the drug can beused to make ribosomes when the drug is removed(56, 91).

Studies of ribosome formation under condi-tions of amino acid deprivation have also beencarried out in HeLa cells. The omission ofmethionine is of special interest, since thisamino acid has a direct role to play in ribosomalprotein synthesis in addition to providing methylgroups for RNA methylation. During methioninestarvation, ribosomal precursor RNA synthesiscontinues, and accumulation of undermethylated45S occurs (99). Cleavage of r-pre-RNA alsocontinues, thereby producing undermethylated32S RNA. No new ribosomal RNA ever reachesthe cytoplasm, however. Also, as was the casewith puromycin treatment, the 18S RNA is soquickly lost that no radioactive 18S RNA is everobserved after uridine labeling of methionine-deprived cells. The undermethylated RNA can be

rescued for processing into ribosomes by therestoration of methionine. Furthermore, it hasbeen demonstrated that undermethylated r-pre-RNA can be methylated during this recovery.

In summary, these experiments on ribosomeformation after puromycin treatment and duringmethionine deprivation focus attention on thecapacity of the animal cell to degrade ribosomalRNA molecules which cannot be properly madeinto ribosomes. Whether such a mechanismexists or is ever used by cells in a tissue is at themoment unknown. However, many tissues whichmake ribosomes only fast enough to replacethose lost by turnover are known to make sub-stantial amounts of r-pre-RNA. Therefore, thepossibility that the rate of ribosome formation isregulated by degradation rather than by process-ing of the r-pre-RNA molecule should be bornein mind.Another recent set of experiments strongly

points to the ability of cultured cells to modulateribosome formation by changing the rate ofr-pre-RNA synthesis. A comparison of the rateof uridine incorporation into 45S RNA ingrowing HeLa cells and in cells deprived ofvaline for longer than 4 hr showed that thestarved cells form 45S less than half as fast as docontrols (99; Maden, Vaughan, Warner, andDamell, unpublished data). In addition, the rate ofmaturation of a 45S molecule, once formed, wasalso slowed down. Thus, the whole machineryfor making ribosomes was synchronously sloweddown. Starvation for a growth-essential aminoacid would be expected to have its effect due tothe limitation of some critical protein, perhaps aribosomal structural protein. It is possible,therefore, that continued work with cultured cellswhich are making ribosomes at different ratescould disclose particular proteins which arecritical in controlling the rate of ribosome syn-thesis.

Such information would not only be veryimportant with regard to control of ribosomesynthesis in normal cells but might also ultimatelybe useful in defining a critical point where controlhas been lost in cancer cells.

PRECURSORS TO TRNA MOLECULES

The role in protein synthesis and the chemis-try of the RNA molecules are far better knownand understood for tRNA than that for othercellular RNA species (46, 60, 114). Very little isknown, however, about the site of tRNA originor whether precursor molecules to it exist. Sincethe major emphasis in this article is on thesynthesis of RNA molecules in animal cells, it isappropriate to describe briefly some results in

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HeLa cells and Ehrlich ascites cells, from whichit appears that a precursor to tRNA has beenfound. One of the difficulties in the past ininvestigating molecules which might be relatedto tRNA was that simple methods for the separa-tion of small RNA molecules of similar size didnot exist. The development of acrylamide gelelectrophoresis of RNA molecules has beenparticularly useful in overcoming this difficulty.For example, the small molecules (5S and 7S)which are part of the larger ribosomal subunitare clearly resolved from 4S RNA by electro-phoresis (55, 66a).

It has been found that HeLa cells which havebeen labeled for 30 min or less contain labeledRNA species which migrate on gel electrophoresisbetween 5S and tRNA (16; D. Bernhardt andJ. E. Darnell, in press). These molecules arelabeled before tRNA becomes labeled and havebeen termed pre-tRNA. If RNA synthesis isstopped by actinomycin in cells where pre-tRNAis the predominantly labeled small RNA species,the pre-tRNA disappears and labeled tRNAappears (Fig. 16). Since the pre-tRNA migratesmore slowly than true tRNA, even in formalde-hyde (Bernhardt and Darnell, in press), a reagentthat destroys secondary structure in polynucleo-tides, it is possible that pre-tRNA is longer thanthe tRNA derived from it.Another finding of interest is that both pre-

tRNA and tRNA formation proceed moreslowly during methionine deprivation. Thissuggests that there may be an involvement of

methylation in the conversion of pre-tRNA totRNA. If this newly described RNA species istruly precursor to tRNA, it should aid con-siderably in locating the cellular origin of tRNA.

DNA-LIKE RNA SPECIESWhile the ribosome and transfer RNA mole-

cules have roles to play in the synthesis of everyprotein, the actual dictation of amino acidsequence is accomplished by the mRNA. Manyevents of interest in animal cells are expressionsof changing patterns of protein synthesis (e.g.,differentiation, cell division, and regeneration),and these must revolve around the availability ofmRNA. Ultimately to understand these processesin molecular terms, it clearly would be desirableto follow mRNA from its synthesis to its utiliza-tion and ultimate destruction. Although mucheffort has been expended in attempting to studymRNA in animal cells, very little concreteknowledge about this RNA species has beenuncovered. One of the outgrowths and one of thesignificant complications of these studies hasbeen the discovery that several classes of cellularRNA exist which share some of the propertiesexpected ofmRNA but which may not be mRNA.This section of the present paper will be con-cerned with this group ofRNA species, which wewill call generically DNA-like RNA.

Definition and Distribution of DNA-like RNASeveral properties of viral and bacterial mRNA

influenced the design of experiments aimed a

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FIG. 16. An apparent precursor to tRNA. A culture ofHeLa cells was labeled with 14C-uridine (solid lines) forone generation and then exposed to 3H-uridine (dashed lines) for 5 min. Actinomycin was added and samples weretaken after 2, 10, 35, and 60 min (A, B, C, and D, respectively). RNA was isolatedfrom cytoplasmic extracts ofeach sample and examined by acrylamide gel electrophoresis for small RNA molecules. The 14C label furnished amarker for the mobility of4S (tRNA) and 5S RNA (Bernhardt and Darnell, unpublished data).

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locating mRNA in animal cells. First, mRNA inbacteria represents a small proportion of thetotal cellular RNA, and it is rapidly synthesizedand degraded (9, 38, 51). Therefore, if bacterialcells are exposed to labeled RNA precursors foronly a brief period, then the amount of radio-activity in mRNA relative to rRNA and tRNA ishigh enough so that mRNA can be studied as aradioactive RNA species. Second, rRNA repre-sents a distinct species of RNA and does nottherefore necessarily reflect the average composi-tion of the total cell DNA; mRNA, on the otherhand, comes from many genes and reflects theaverage DNA composition (92).One final property of mRNA needs emphasis.

Although distinct from rRNA, it must be as-sociated with ribosomes, at least during the timeit is directing protein synthesis (9).

It was previously pointed out (Fig. 4) that thetotal labeled RNA from animal cells exposed to32p for 30 to 40 min contained a large propor-tion of labeled molecules which sedimented morerapidly than 45S r-pre-RNA. When the basecomposition of the large molecules was deter-mined, it was found that they resembled DNA,i.e., had a GC content of 43 to 47% (la, 24, 87,90, 112). Thus, this material was potentiallymRNA. However, as previously mentioned, mostof the rapidly labeled RNA of total cells wasknown to be in the cell nucleus. This largeheterogeneously sedimenting DNA-like RNA,termed HnRNA, also has been shown to exist inthe nucleus. Recent cell fractionation studiesshow that the HnRNA is largely, if not com-pletely, unassociated with the nucleolus (107; S.Penman, C. Vesco, and M. Penman, J. Mol. Biol.,in press). The HnRNA therefore may representwhat has been termed by cytologists "chromo-somal RNA" (23).

If cytoplasmic extracts of briefly labeled cellsare examined, it is found that about 5 to 10% ofthe total incorporated radioactivity is cytoplasmicrapidly labeled RNA (107). Examination of thedistribution of rapidly labeled cytoplasmic RNAshows that, although most of the radioactivity isin the so-called soluble fraction (i.e., nonsedi-mentable in 1 to 2 hr at 100,000 X g), there islabeled RNA associated with structures of allsizes from 40 to 300S (Fig. 17; 57). In the poly-ribosomes of cells labeled with 3H-uridine for 30min or less, labeled RNA can be recoveredfree from protein with the sedimentation pro-file shown in Fig. 17. It will be immediatelyrecognized that this polysome-associated rapidlylabeled RNA (i) sediments more slowly than theHnRNA and (ii) does not conform to the sedi-mentation pattern of rRNA or tRNA. In addi-

0J

N x0-u

II

FIG. 17. Distribution of radioactivity in cytoplasmicextracts of briefly labeled HeLa cells. Left panel:cytoplasmic extracts of HeLa cells labeled with 3H-uridine were sedimented in sucrose gradients (condi-tions as for center panel, Fig. 11) and assayed foracid-precipitable radioactivity. Right panel: polysomal-associated RNA was further examined (by releasingthis RNA with sodium dodecylsulfate and examiningit on a sucrose gradient. Redrawn from Latham andDarnell (57).

tion, it has been found that the base compositionof this polysomal RNA is very different fromtRNA or rRNA and is more similar to cellularDNA (Table 1). This rapidly labeled DNA-likeRNA species from polyribosomes has beenconsidered to represent mRNA. Several addi-tional lines of experimentation indicate that thisinterpretation is correct. (i) The cytoplasmicextracts of cells infected with various animalviruses have been shown to contain virus-specificRNA complexed with host ribosomes in viralpolyribosomes (3, 68, 82, 96). Such a result isdiagrammed in Fig. 18, where it is shown thatwhole molecules of poliovirus RNA are recovera-ble from poliovirus polyribosomes. An additionalpoint of importance comes from this experiment-since whole viral RNA molecules are found invirus polyribosomes, it is likely that mRNAmolecules, cellular or viral, obtained frompolyribosomes of HeLa cells are not degraded.(ii) The chemical stability of polyribosomessuggests a complex between ribosomes andmRNA. For example, the chelating agentethylenediaminetetraacetate destroys polyribo-somes (34) and releases most of the rapidlylabeled RNA from the polysome region of asucrose gradient (Fig. 19). (iii) Puromycin isknown to cause the discharge of the nascentpolypeptide chain from ribosomes of mRNA,and polyribosomes are thereby degraded bypuromycin treatment. If cells which have beenbriefly labeled are treated with puromycin, verylittle of the rapidly labeled cytoplasmic RNA isfound in the polysome region (Fig. 19).

In summary, the species of rapidly labeled

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cytoplasmic RNA considered to be mRNA isthat which is associated with polyribosomes and Acan be discharged by ethylenediaminetetraacetateor puromycin treatment. An extensive study onthe time course of synthesis and appearance of -mRNA employing these criteria for mRNA hasjust been completed (S. Penman, C. Vesco, and --- EDTA P-- romycinM. Penman, J. Mol. Biol., in press).Perhaps the two most important questions

which arise from present experiments on DNA-like RNA species are the following. (i) Does therapidly labeled RNA found in cytoplasmic /structures which sediment at 40 to 100S before

T~~~~~~~~~~~~~~~~~~

5000 0IB|^{@I_5000 5 Polysomee 74S uuni3s Polysomes 74SSuLuni+s

FIG. 19. Effect of EDTA and puromycin treatmenton the radioactivity in polyribosomes. Left panel:

asU . HeLa cells were labeled for 25 min with 3H-uridine,1000 - 0.5 and sedimentation of the total acid-precipitable radio-> l \ ff O activity was examined in hypotonic buffer containingcL / \ ^ \ P 0.0015 M MgCI2 (RSB) or EDTA (0.01 M). ConditionsI\\a, of sedimentation were similar to those shown in right-

500 I hand panel of Fig. 11 (7 to 47% sucrose gradients).Only radioactivity profiles are shown. Regions of gradi-ents are marked according to OD260 of RSB gradient.Right panel: HeLa cells were labeled for 20 min with

________ 3H-uridine and the culture was divided. One half wasI. A I. B continued at 37 C for 5 additional mini, and the other

2,S 22S half was treated with 200 ,ug/ml of puromycin duringthe last 5 min of incubation.' Cytoplasmic extracts werethen made and analyzed as described above (data of C.Birnboim and J. E. Darnell, unpublished).

deproteinization represent mRNA on the way tobecoming polyribosomes? (ii) What is the

o000 relationship, if any, between HnRNA andpolysomal mRNA?

Several results argue against the possibility thatthe rapidly labeled RNA recovered from the 40to lOOS structure found in cytoplasmic extractsis new mRNA enroute to polyribosomes. (i) Ifcells are treated with actinomycin, thus stoppingRNA synthesis but leaving protein synthesis

20 20 unaffected for several hours, the labeled RNAFIG. 18. RNA from poliovirus polyribosomes. HeLa from the 40 to 100S region does not "chase" into

cells which had been grown in 14C-uridine were infected polysomes (57). (ii) It was found with L cellswith poliovirus and treated with actinomycin to stop that the manner in which cytoplasmic extractshost-cell RNA synthesis. The culture was labeled with were prepared determined how much label was3H-uridine throughout virus infection. Viral polyribo- found in the 40 to 100S region. Thus, if cellularsomes were isolated by sedimentation of cytoplasmic extracts of cells which were broken in isotonicextracts (top panel, A and B are polyribosome frac- medium containing a nonionic detergent weretions; S is single ribosomes; solid line, optical density compared to extracts prepared by the moreat 260 nm) and RNA from these was released and compae toetcts ard by nth orefurther examined by sucrose gradient analysis (lower commonhypotomcswelltg and homogenization,panel). Viral RNA is 3H-labeled (0) and host cell itwas found that the yield of polysomes wasRNA is 14C-labeled (0). From D. F. Summers, J. V. equivalent but the amount of rapidly labeledMaizel, and J. E. Darnell, (Virology 31:427, 1967). RNA in the 40 to 100S region was decreased by a

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factor of 10 in the isotonically prepared extracts(Perry and Kelley, J. Mol. Biol., in press). Theseresults suggest that a large fraction of the 40 tolOOS material might derive from the nucleus.While the exact nature of the labeled RNA in the40 to lOOS region is not clear at present, theseresults suggest that it may bear no relation tomRNA.

In order to adequately discuss the possiblerelation ofmRNA to HnRNA, we must describeexperiments on the HnRNA in more detail.

HnRNA: Properties and Problems

The very rapidly sedimenting (20 to lOOS)HnRNA which we wish to consider in somedetail has been found not only in the nuclei ofcultured cells but in many differentiated cells aswell. Work in one of these systems, nucleatedduck erythroblasts, has provided good evidenceabout the eventual fate of the HnRNA found inthose cells. Because the duck erythroblast, ahighly differentiated cell making primarilyhemoglobin, makes few if any new ribosomes, itwas possible to label the cells with an RNAprecursor, remove the label, and study whathappens to the previously labeled HnRNA as afunction of time (la, 87). Experiments of this typedefinitely show that the majority of the HnRNAis not transferred to the cytoplasm even hoursafter its synthesis, and 60 to 70% of the totalradioactivity in RNA is gradually lost, presuma-bly back to the acid-soluble pool. If after thelabeling of HnRNA, either in duck erythroblastsor cultured cells, the cells are treated withactinomycin D, the great majority (about 90%)of the HnRNA decays to acid-soluble material(48, 107). It was strongly suggested therefore that,at least in the duck erythroblast, the majority ofthe HnRNA never serves a cytoplasmic function(1, 87).To investigate this idea more thoroughly in

growing cultured cells, an attempt has been madeto quantitate the various fractions of the cellinvolved and to determine whether the amount oflabel found in various fractions was consistentwith a constant nuclear turnover of the HnRNAor whether it was synthesized at a rate consistentwith its utilization as cytoplasmic mRNA (Table1; Soeiro et al., in press).The cytoplasmic poiyribosomes of HeLa cells

constitute the majority of the cell's ribosomes,and the average size is about six ribosomes perchain of mRNA, the average size of which isabout 12 to 16S or approximately 6 x 105. Thus,the ratio of rRNA to mRNA in these structuresis at least 20:1 [6 x (1.7 x 106 + .6 x 106)/6 x 105]. If both the rRNA and mRNA were

stable, then the cell would have to synthesize 20times as much RNA which was precursor toribosomes as that which was precursor to mRNA.The mRNA, however, is thought to be an un-stable molecule (relative to the rRNA) with ahalf-life of about 3 to 4 hr at the shortest (69).Taking the minimum ratio of rRNA to mRNAof 20:1, and the shortest possible half-life formRNA, it was calculated that the cell shouldstill make at least three times as much rRNA asthat which was precursor to cytoplasmic InRNA(90, 107). Actually, since we now know thatapproximately 50% of the r-pre-RNA moleculeis itself synthesized and degraded and not used inthe final ribosomal product, we would expect sixtimes as much 45S r-pre-RNA as any DNA-likeRNA which was precursor to cytoplasmicmRNA.With these thoughts in mind, a careful assess-

ment of the relative amounts of radioactivity in45S r-pre-RNA and HnRNA was made bylabeling cells with 32p and analyzing the RNA byzonal sedimentation followed by base analysis.Because the average GC content of HnRNA islow, about 44%, and that of highly purified 45Shad been determined to be 70% GC (Table 1;1, 52), it was possible to determine accurately thedistribution between these two classes of RNAin the total rapidly labeled nuclear RNA (Soeiroet al., J. Cell Biol., in press).The results shown in Fig. 20 and Table 4

indicate that the 45S r-pre-RNA does not con-stitute the bulk of rapidly labeled material, buton the contrary the HnRNA represents 75% ormore of the nuclear radioactivity after 10- or20-min exposure to 32p. Furthermore, the shorterthe label time the higher the proportion ofradioactivity in the HnRNA. The flow of radio-activity is much faster into HnRNA than intor-pre-RNA, although they constitute almost thesame total amount of RNA. As pointed outpreviously, any precursor to cytoplasmic mRNAshould be synthesized no faster than aboutone-sixth the rate of synthesis of r-pre-RNA.The results described above prove that most

of the HnRNA turns over in the cell nucleus, anidea first proposed a number of years ago byHarris (42).While it is clear from the foregoing discussion

that most of the HnRNA does not eventuallybecome cytoplasmic mRNA, it is possible that asmall portion does. Several explanations mightbe suggested, all of which involve a scission ofthe HnRNA with partial use of the products asmRNA. (i) The HnRNA has a much highermolecular weight (electron micrographs indicate

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that itmay be as long as 10 ,um orabout 107 daltons;88) than cytoplasmic mRNA, and a small portionof each molecule could serve as mRNA. (ii)Many extra copies of each HnRNA moleculemight be made and only a few of each typeeventually used to generate mRNA. (iii) Most ofthe HnRNA might represent types of moleculeswhich are never used even in part, but a smallfraction of the total number ofHnRNA moleculesmight be used wholly or in part.Of course, it should again be pointed out that

it is possible that none of these conjectures iscorrect and that the HnRNA bears no relation-ship to the cytoplasmic mRNA but is entirelydestroyed within the nucleus.These crucial questions cannot be answered by

kinetic experiments alone. Some means ofcomparing the sequences of HnRNA and thevarious cytoplasmic DNA-like molecules isnecessary. As pointed out in the first section ofthis paper, at present the only feasible approachto sequence relatedness among RNA moleculesis hybridization of RNA to DNA. Because thistechnique has the potential of answering thisquestion, experiments on the hybridization ofDNA-like RNA from cultured cells will bediscussed in a separate section.

0Hx

(+±P

A 23.2 22.8 19.2 19.7 22.4G 19.9 19.9 24.1 24.5 22.0U 32.5 32.6 282 29.0 24.1

FIG. 20. Base composition analysis of RNA labeledin 10 min by 'P. Total nuclear RNA was isolatedfromHeLa cells after a 10-min exposure to 32p and sub-

jected to sucrose gradient separation. Base compositionanalysis of RNA from various sections of the sucrose

gradient was then performed. Bar graph indicates per-centage GC obtained. From Soeiro, Vaughan, Warner,and Darnell (in press).

TABLE 4. Relative amounts of HnRNA and r-pre-RNA from briefly labeled cellsa

Length Sedimenta-of label tion values(i) of nuclear(m) RNA

10

20

77- 115545-77S45537-45S10-37STotal

77-1lSS45-77S45S40-45537-40S10-37STotal

Basecomposi-

tion(% GC)

434351.45049

414454534754

Totalcount/min

3146463240

105190120918680

Calculated counts/min as

HnRNA r-Pre-

314631 1522 1030 10160 -35

10519072617455

557

48301225

115

a 32P-labeled RNA from experiment shown inFig. 20 was analyzed for base composition andamount of RNA of each major class (HnRNA,44% GC; r-pre-RNA, 70% GC) estimated.

Use of RNA-DNA Hybridization in the Study ofDNA-like RNA Species

The original demonstration of RNA-DNAhybridization was with mRNA from bacterio-phage-infected cells and bacteriophage DNA (41,93). This was soon extended to bacterial RNAand DNA. We wish to consider how the tech-niques used in these studies and the resultsobtained can help design hybridization studieswith animal cell DNA-like RNA and the DNAfrom which it came.

First, it is worthwhile to mention that twobasic methods of allowing the RNA to interact(anneal) with the DNA have been employed. (i)The annealing procedure can be carried out bymixing the RNA and DNA in liquid at an ele-vated temperature and a chosen salt concentration(41, 93). One disadvantage of this technique hasbeen pointed out, namely, the opportunity forDNA-DNA reannealing to proceed and therebyforeclose the opportunity for formation ofRNA-DNA hybrids. A second disadvantage,which will be emphasized in later discussions, isthat, since the reactants are present in solution,mixtures cannot be easily washed free from onetype of RNA in order to re-expose the DNA to asecond type of RNA. (ii) Another means ofconducting hybridization is to fix the single-stranded DNA to a solid substrate (2, 7, 33)prior to exposure to RNA. Two advantages ofsuch techniques exist. Not only is the fixed DNA

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prevented from reannealing, but it also becomespossible to expose the DNA to one RNA sample,wash this away, and introduce a second RNAsample. As will be described in detail, the fixationofDNA to nitrocellulose filters (33) is of particu-lar value in doing competition experiments withdifferent RNA samples.With bacteriophage mRNA and the various

bacterial RNA species, two general types ofresults have been attained. The first type ofresult is of qualitative or diagnostic value. Forexample, the rapidly labeled RNA is isolatedfrom a small culture of bacteria or bacteriainfected with T-even bacteriophage and thelabeled RNA is exposed to bacterial or phageDNA (41, 93). In the case of labeled RNA fromgrowing cells, a large fraction of the addedradioactive RNA will become bound to bacterialDNA but none will bind to phage DNA. Thelabeled RNA from phage-infected cells con-versely will bind to phage and not bacterial DNA.Two important points emerge from experimentsof this type. First, the hybridization reaction hasspecificity and, second, it is implied, especiallyfrom the experiments with bacterial RNA, that abroad segment of the genome is represented inthe labeled RNA because a large amount (10 to50%) hybridizes. If the labeled bacterial RNAused in the hybridization reaction is purifiedrRNA, only a very small fraction of the input(1 % or less) is found to hybridize. Thus, readilyhybridizable RNA is often equated with RNAdrawn from a broad segment of the genome ofthe cell in question.The introduction of quantitation into hy-

bridization experiments came with the hybridiza-tion of purified bacterial rRNA to bacterial DNA(41, 93). In this type of experiment, increasingamounts of labeled rRNA were mixed with afixed amount of DNA. Ultimately, a point wasreached where continued addition of labeledrRNA did not result in the formation of addi-tional rRNA-DNA hybrid. Since the specificactivity (counts per min per ,ug of rRNA) of therRNA and the amount of DNA employed inthese experiments was known, the amount ofrRNA which could bind to a given amount ofDNA could be calculated. This saturation valueof Escherichia coli DNA for rRNA has beenfound to be 0.3 %; i.e., 0.3 % of the E. coli DNArepresents sites from which rRNA could be pro-duced. As was pointed out earlier in this re-view, this "saturation" technique has foundgreat use in ascertaining the percentage of me-tazoon genomes responsible for rRNA, 5S RNA,and tRNA (11, 12).Another type of hybridization experiment

which is based on the fact that the DNA sites canbe saturated is the so-called "competition"hybridization experiment. The first example ofthis type of experiment to be described involvedthe extraction of unlabeled or labeled RNA fromT4 bacteriophage-infected cells early and lateduring the phage replicative cycle (40). A quali-tative difference in the types of mRNA moleculesof the "early" and "late" type was inferred fromthe finding that the hybridization of radioactivelylabeled "early" or "late" mRNA could bereduced most effectively by the addition to thehybridization mixture of homologous unlabeledmRNA. An important modification of the basiccompetition technique has recently been de-scribed (54). Because the RNA-DNA hybridonce formed at a given temperature and saltconcentration is a stable structure under theconditions of its formation, Kasai and Bautzexposed phage DNA (bound to nitrocellulosefilters) to unlabeled competing RNA (early orlate phage mRNA) and then washed the filtersfree from any unbound RNA. Upon subsequentchallenge of the filters with labeled RNA, it wasfound that the sites on the DNA which wouldhave accepted labeled RNA had been pre-saturated by the unlabeled competing RNA.The DNA on the filters was still capable ofreacting to heterologous phage mRNA.Obviously, this stable preoccupation of all of acertain type of sites on DNA implies that asaturating amount of unlabeled phage mRNAwas employed in these experiments.

Hybridization of DNA-like animal cell RNAto its homologous DNA has been demonstratedin many instances (5, 49, 86, 111). A great manyrecent attempts to achieve saturation conditionsand perform competition hybridization have alsobeen reported. In almost all of these competitionexperiments, labeled RNA and "competing"unlabeled RNA have been simultaneouslyexposed to DNA. A decrease in the amount oflabeled RNA-DNA hybrid has been interpretedto mean that sites on the DNA which wouldhave been occupied by labeled molecules hadbeen pre-empted by unlabeled molecules. Insupport of this, it has been pointed out thattotally unrelated RNA from bacterial or yeastcells does not prevent the animal cell RNA-DNAhybrids. What has not been demonstrated in anyof these experiments is that the decrease inhybrid formed is due to a specific occupation ofthe DNA sites by the competing unlabeled RNA.In other words, the Kasai-Bautz presaturationtechnique has not been employed.From recent experiments involving relatively

large amounts of purified HnRNA from HeLa

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cells, it appears that true saturation of the DNAby this species of RNA has been achieved andthat true competition between unlabeled HnRNAand labeled HnRNA can be demonstrated byfirst exposing DNA to unlabeled HnRNA,washing the DNA-bearing filter and re-exposingto labeled RNA (Soeiro and Darnell, unpublisheddata). With 1 ,ug of HeLa cell DNA attached to aMillipore filter, between 0.025 and 0.05 ggof HnRNA can be bound. (Conditions employedwere 65 C, 20 hr of incubation in 0.3 M NaCl,0.03 M sodium citrate, and 0.1 % sodium dodecylsulfate.) Saturation of the DNA on the filter isnot achieved, however, with inputs of less thanabout 20 ug of HnRNA, a 400-fold excesscompared to what is bound. In this connection,it is pertinent to point out that a number ofreports exist in tihe literature of studies in whichsaturating amounts of rRNA and also tRNAand 5S RNA have been determined (61a, 80). Inall cases, a ratio of input RNA molecules toavailable DNA sites of at least 100 was necessaryto achieve saturation.

If the sites on HeLa cell DNA can be saturatedby HnRNA, then pre-exposure of the 1-,g HeLaDNA filters to unlabeled HnRNA in the rangeof 20 ,g should result in the saturation of theDNA sites for HnRNA by stable hybrid mole-cules withl unlabeled HnRNA. Subsequentreaction of the DNA with labeled HnRNAshould be blocked. Such a competition experi-ment carried out by presaturation of the DNA isshown in Fig. 21 (left panel). Most of the sites onthe DNA can obviously be occupied by stablehybrids of unlabeled HnRNA.

Also shown in Fig. 21 is the result of simul-taneously exposing HeLa DNA to labeled andunlabeled HnRNA molecules. It can be seen thatsimultaneous exposure to hot and cold HnRNAcauses a depression in the amount of labeledRNA-DNA hybrid formed which is greater thanthat observed by presaturation. Even at RNAinputs substantially below the saturation levelfor the 1 4g DNA filters, there is considerableinterruption of hybrid formation. This interrup-tion cannot be due to stable hybrid formation ofunlabeled HnRNA with a DNA site which mightotherwise be occupied by a labeled moleculebecause of the results obtained in the presatura-tion experiments.

Presaturation of DNA would appear to be themethod of choice to demonstrate true competitionbetween unlabeled and labeled samples of RNA.A final point in support of this conclusion can beseen in Fig. 21 (right panel) where it is shown that,although HeLa cell DNA can be presaturated bycold HnRNA, L-cell DNA cannot be so blocked.

0\\

\ \ HELA VS L-CELL0 50 a

Z 0

0

20 20MICROGRAMS COMPETING RNA

FIG. 21. Competitiont hybridization of HiiRNA.Left pantiel: radioactive HnRNA froni HeLai cells washybridized with HeLa cell DNA (I ug, bolunid to Milli-pore filters; 33), and/ the effiect of itoiltabeled HeLa cellHutRNA oni this process was stludied. Increasing amoititsof unlaibeledl HniRNA were dcldledl durinig the hybridiza-tioni oJ the hot RNA ('simnultaneous competition,"filled sYmbols) or tile iulaibeledl HniRNA was acddiedprioi. to the labelel RNA, catid ioihoiind RNA wasemnoved be/ore the DNA wvas exvposed to labeled RNA("presaturation," open symbols). Diff,,cerlt symbolsrepresenit dilferenit preparationls oflunllcabeled RNA. Thelevel of hybr-idized Icbeledc RNA without competinigunlabeled RNA ranged fr11m 400 to 1.000 coltuitslinin.Right pcaniel: radioactive HniRNA .1/romii L cells wasplirified anid hybridized to L-cell DNA. The effect ofHeLca-cell Hn1RNA oni the L-cell hiybridization wacsstutdiecd bw 'simultaneous' adl 'presatulration"'' comn-petitionl techniques.

However, simultaneous addition of unlabeledHeLa HnRNA depresses both HeLa and L-cellRNA-DNA hybrids.

Using the presaturation method, it may bepossible to obtain more meaningful informationabout sequence similarities among moleculeswhich are similar in overall composition butwhich come from different cellular locations.

CONCLUSIONS

In an active field of research, an article at-tempting to summarize available knowledge isdoomed to obsolescence by press time because ofthe rapid accumulation of new information.Nevertheless, it seems worthwhile to pause atleast momentarily to list the positive statementsabout animal-cell RNA metabolism which havecome from the work of the past few years as wellas to focus as clearly as possible on those areaswhich need a great deal more illumination.On the positive side, it can be stated that the

manufacture of ribosomes in animal cells isbegun in the nucleolus by the transcription of ahigh molecular weight precursor to rRNA. Thismolecule is methylated and becomes associatedwith ribosomal protein within a brief time after

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its synthesis. It is cleaved at specific sites so thattwo ribosomal subunits, one containing 18SrRNA and the other 28S rRNA, plus two smallRNA chains eventually emerge. A substantialportion of the ribosomal precursor RNA mole-cule is lost in the maturation process.To choose one example of the kind of problem

which awaits solution in the area of ribosomeformation, one can mention the presumablyenzymatic scission of the rRNA precursor.Isolation of such enzymes and extensive study ofthis process should contribute greatly to anunderstanding of how specific protein-nucleicacid interactions are mediated.

In addition to ribosome biogenesis, thisarticle has dealt with various types of animal cellRNA molecules which resemble DNA in theiroverall base composition. Here, we have pro-gressed only far enough to distinguish varioustypes of this DNA-like RNA on operationalgrounds as mRNA, cytoplasmic DNA-like RNA,and HnRNA. As was heavily stressed, a problemof commanding importance is to understandwhat, if any, relationship these various types ofDNA-like RNA molecules have to one another.

Finally, as a prime example of a whole body ofnew information which is just around the corner(but, fortunately for this reviewer, enough in thefuture to have just missed this report), onecould mention the RNA associated with mito-chondria. It is possible that mitochondria notonly generate some mRNA from their DNA butalso some ribosomes may originate in mito-chondria. It is conceivable that some of theknottier problems of animal cell biology such asmRNA generation may not be solved by a studyof nuclear function but by dealing with mito-chondria.At any rate, the general subject of gene expres-

sion in animal cells as approached by examinationof animal cell RNA is progressing rapidly andcan undoubtedly be profitably reviewed againsoon.

ACKNOWLEDGMENTSThe work described in this paper from the author's

laboratory was supported by grants from the NationalInstitutes of Health (CA 07861-04), the NationalScience Foundation (GB 4565), and the HealthResearch Council of the City of New York.The author also thanks Sheldon Penman, Jonathan

Warner, and Eric Holtzman for making availablevarious prints, and H. C. Birnboim and DeborahBernhardt for making available some unpublisheddata.

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la. Attardi, G., H. Pamas, M.-L. H. Hwang, andB. Attardi. 1966. Giant size rapidly labelednuclear ribonucleic acid and cytoplasmicmessenger ribonucleic acid in immature duckerythrocytes. J. Mol. Biol. 20:145-182.

2. Bautz, E. K. F., and B. D. Hall. 1962. The isola-tion of T-4 specific RNA on a DNA-cellulosecolumn. Proc. Natl. Acad. Sci. U.S. 48:400-408.

3. Becker, Y., and W. K. Joklik. 1964. MessengerRNA in cells infected with vaccinia virus.Proc. Natl. Acad. Sci. U.S. 51:577-585.

4. Bernhard, W., and N. Granboulan. 1963. Thefine structure of the cancer cell nucleus. Exptl.Cell Res. Suppl. 9:19-53.

5. Birnboim, H. C., J. J. Pene, and J. E. Darnell.1967. Studies on HeLa cell nuclear DNA-likeRNA by RNA-DNA hybridization. Proc.Natl. Acad. Sci. U.S. 58:320-327.

6. Blobel, G., and V. R. Potter. 1966. Nuclei fromrat liver: isolation method that combinespurity with high yield. Science 154:1662-1665.

7. Bolton, E. T., and B. J. MacCarthy. 1962. Ageneral method for the isolation of RNAcomplementary to DNA. Proc. Natl. Acad.Sci. U.S. 48:1390.

8. Brachet, J. 1940. Histochemical detection ofpentose nucleic acid. Compt. Rend. Soc.Biol. 133:88.

9. Brenner, S., F. Jacob, and M. Meselson. 1961.An unstable intermediate carrying informa-tion from genes to ribosomes for proteinsynthesis. Nature 190:576-581.

10. Britten, R. J., and R. B. Roberts. 1960. Highresolation density gradient sedimentationanalysis. Science 131:32-33.

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