THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

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1 THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS MARGHERITA WILLEMS, MARIA PENMAN, SHELDON PENMAN and Frm the Massachusetts Institute f Technlgy, Cambridge, Massachusetts ABSTRACT INTRODUCTION The effect f prtein synthesis inhibitin by cyclheximide n nuclelar RNA synthesis and prcessing has been studied in HeLa cells. Synthesis f 45S RNA precursr falls rapidly after administratin f the drug. Hwever, the nuclelar cntent f 45S RNA remains relatively cnstant fr at least I hr because the time required fr cleavage f the precursr mlecule int its prducts is lengthened after treatment with cyclheximide. The efficiency f transfrmatin f 45S RNA t 32S RNA remains cnstant with apprximately ne mlecule f the 32S RNA prduced fr each cleavage f a mlecule f 45S RNA. Hwever, shrtly after the cessatin f prtein synthesis the frmatin f 18S RNA becmes abrtive. The amunt f 32S RNA present in the nuclelus remains relatively cnstant. After lng perids f prtein synthesis inhibitin the 28S RNA cntinues t be synthesized and exprted t the cytplasm but at a greatly reduced rate. When the prtein synthesis inhibitr is remved, a prmpt, althugh partial, recvery in the synthesis rate f 45S RNA ccurs. The varius aspects f RNA synthesis regulatin and prcessing are discussed. The nuclelus is a highly structured and specialized rganelle. Its principal and perhaps nly functin is the prductin f ribsmes (I-4). The regulatin f ribsmal precursr RNA synthesis and prcessing under cnditins f prtein synthesis inhibitin is the subject f this reprt. It has been shwn previusly by Warner that the structural prteins fr ribsmes can be fund assciated with the nuclelus (5). Furthermre, a pl f these structural prteins must exist, since under cnditins f prtein synthesis inhibitin newly labeled ribsmal RNA can be detected in the functining ribsmes in the cytplasm (6). Previus studies f nuclelar functin after the inhibitin f prtein synthesis have established the fllwing pints. The nuclelus can still prduce mature ribsmes, althugh at a reduced rate (6). Hwever, the frmatin f 18S RNA frm 45S precursr ceases sn after inhibitin (7, 8). Since 32S and 28S RNA cntinue t be frmed, it is apparent that 18S RNA is degraded under these cnditins. Seir, Vaughan, and Darnell have shwn that the time fr prcessing 45S ribsmal precursr is increased during the inhibitin f prtein synthesis (8). They als established that the prductin f aberrant plypeptides in purmycin-treated cells results in further interference 1 7 7

2 with nuclelar functin s that n cytplasmic 28S RNA is btained. In this reprt, a detailed study f nuclelar f>nctin during the inhibitin f cellular prtein synthesis is described. The respnse f the nuclelus appears t be regulated. The rate f 45S ribsmal precursr falls rapidly in the presence f the prtein synthesis inhibitr, cyclheximide. Hwever, the nuclelar cntent f 45S and 32S RNA changes nly slightly. This decrease in the rate f synthesis withut a cncmitant decrease in the nuclelar cntent f RNA is shwn t be the result f an increased length f time required fr RNA prcessing. Thus, synthesis and prcessing time are interrelated in such a way as t maintain a relatively cnstant nuclelar cntent f RNA. Even thugh nuclelar functin is drastically altered during prtein synthesis inhibitin, a cnstant efficiency f transfrmatin f 45S t 32S is maintained. Hwever, as shwn previusly, prductin f 18S RNA rapidly ceases after prtein synthesis inhibitin. Presumably, the inhibitin f synthesis f structural prteins results in this species being degraded after it is cleaved frm the 45S precursr. MATERIALS AND METHODS The experimental results presented here can be best understd in terms f results f previus investigatins which utlined the details f RNA prcessing in the nuclelus (15), which are illustrated in Fig. 1. The initial event in the frmatin f ribsmal RNA is the synthesis and methylatin f a larger precursr mlecule whse sedimentatin is 45S. The precursr remains as 45S fr abut 16 min and then is cleaved t 18S and 32S RNA. The prcess f cleaving the precursr mlecule t frm its prducts is nt cnservative because the cmbined mlecular weights f the 18S and 32S species f RNA are much less than the mlecular weight f the 45S pre-. The experiments were perfrmed with suspensin cultures f S-3 HeLa cells grwing in Eagle's medium supplemented with 7% hrse serum ata nminal cncentratin f 4 X 105 cells/ml (9). Cyclheximide was used at a cncentratin f 100 µg/ml, except when specified. Befre additin t the culture medium f radiactive RNA precursrs, the cells were cncentrated t 2 X 106 cells/ml. The preparatin f nuclelar, nucleplasmic, and cytplasmic fractins was as described (2) except that the nucleli were separated frm DNase-digested nuclei by znal centrifugatin thrugh a sucrse density gradient (10). SDS-phenl extractin f the RNA in the three cellular fractins was as described (11), except that the temperature f extractin was 55 C (12). The purified cursr (10, 15, 16,1 ). RNA was analyzed by SDS-sucrse gradients (13) r by acrylamide gel electrphresis (14, 15). The cmpsitin f the buffer used in SDS sucrse gradients was the fllwing : 0.5% sdium lauryl sulfate (SDS), 0.1 M NaCI, 0.01 M Tris, ph 7.4, M EDTA. Cyclheximide (Acti-dine) was purchased frm varius species (16). Calbichem, Ls Angeles, Calif. Electrphretically purified DNase was btained frm Wrthingtn Bichemical Crp., Freehld, N. J. Actinmycin was a gift f Merck, Sharp & Dhme I McCnkey, E. Private cmmunicatin. 45S CYTOPLASM Wv CYTOPLASM FIGURE 1 Schematic representatin f the stages in prcessing f the 45S RNA nuclelar precursr t ribsmes. Uridine-2-14C (27 mc/mmle) and uridine-5 3H (20 me/µmle) were purchased frm Schwarz Bi Research Inc., Orangeburg, N. Y. RESULTS Evidence fr the lss f material during the nrmal prcessing f 45S has been btained by studying methyl t uridine ratis f the varius species f RNA (10, 15), the base cmpsitins f precursrs and prducts (10, 16), and the pancreatic RNase digests f the All these techniques pint t a disappearance f mre than 30% f the 45S mlecule during the frmatin f 18S and 32S TnF JOURNAL OF CEId. BIOLOGY. VOLUME 41, 1969

3 a. b. I I I 15 J O tr H 100 z ô prducts. A clearly bserved transient 41S species and a minr 36S species f nuclelar RNA have been identified and are presumably intermediates in the cleavage f the 45S precursr (15). The 18S mlecule is rapidly exprted t the cytplasm where it appears in the small ribsmal subunit. The 32S mlecule remains intact in the nuclelus fr a minimum f 40 min (2), after which time it is cleaved again t frm the 28S species f RNA. This cnversin in sedimentatin has been demnstrated t be a mlecular weight change with apprximately 30% f the 32S mlecule being lst (10, 15, 16). The evidence that 28S RNA is lwer in mlecular weight than 32S RNA cmes frm the study f methyl t uridine ratis f the tw species (15), the base cmpsitin (10), the cmparisn f electrphs) O - x 10 a 50 CYTOPLASM 5 \ N UCLEOPLASM i NUCLEOLUS I TIME (MIN) FRACTION FIGURE 2 10s cells were suspended in 50 ml f medium and divided as fllws. 2 X 10 7 cells (cntrl) were labeled fr 10 min with uridine-5-3h (5µC/ml ; sp. act. 25 me/µmle). Incrpratin was terminated by pipetting the cells nt crushed frzen Earle's saline. Equal aliquts f cells were treated with cyclheximide fr 5, 30, 60, and 120 min, and then labeled as abve. Nucleli were prepared and RNA was extracted as described in Materials and Methds. The cytplasmic and nucleplasmic fractins were made 0.5% in SDS (sdium lauryl sulfate) and an aliqut f each was precipitated with 10% trichlracetic acid, cllected n a Millipre filter (Millipre Filter Crp., Bedfrd, Mass.), and cunted in an Ansitrn liquid scintillatin cunter. The extracted nuclelar RNA was analyzed n 2.6% plyacrylamide gels as described in Materials and Methds. Gels were run fr 4.5 hr at 5 ma per gel. Optical density (OD) was mnitred with a specially adapted Gilfrd recrding spectrphtmeter (Gilfrd Instrument Cmpany, Oberlin, Ohi). The gels were then cut int I min lengths, hydrlyzed with 0.4 ml f NH40H, and cunted in a scintillatin cunter. The ttal radiactivity in the 45S regin f the nuclelar gel was calculated and pltted as % cntrl in Fig. 2a. Cytplasmic and nucleplasmic radiactivity was graphed in the same manner. Fig. 2b shws the prfile f radiactivity in the nuclelar gels in the cntrl - and after treatment with actidine fr 5 min - O- O- and 60 min A---A---A. retic mbility t sedimentatin velcity (15), and the pancreatic RNase digest (16). The 28S RNA cleaved frm the 32S remains in the nuclelus r nucleplasm fr anther 20 min befre emerging int the cytplasm. An apparent nucleplasmic stage fr this 28S RNA has been previusly reprted (2, 17), but this may represent, at least in part, RNA which is assciated with the nuclelus in viv and is brken lse by the high-salt treatment used t prepare the nuclelus (18). It shuld be nted that the lifetimes f the varius species f RNA are nminal values and that there is cnsiderable dispersin in the time it takes fr individual mlecules t transit these stages. Fig. 2a shws the decline in the rate f labeling f varius species f HeLa cell RNA after the administratin f cyclheximide. Cells were labeled 30 WILLEMS ET AL. RNA Synthesis and Prcessing during Prtein Synthesis Inhibitin 1 7 9

4 OD 260 C 30 MIN CM CM OD 1 ^ I I MIN 100 Z L- I CM TIME (MIN) FIGURE X 108 cells were suspended in 60 ml f medium and labeled fr 60 min with uridine-2-14 C (0.1 µc/ml ; sp. act. 50 inc/mmle) which was diluted with 0.15 mg f unlabeled uridine. An aliqut f 2 X 10 7 cells was taken (cntrl) and the remaining culture was treated with cyclheximide. Samples were taken at 10, 25, 40, 60, and 90 min after additin f Acti-dine. Nucleli were prepared and RNA was extracted as described in Materials and Methds. Nuclelar RNA was analyzed n plyacrylamide gels as in Fig. 2. Ttal radiactivity in the 45S RNA was calculated and pltted as % cntrl. fr 10 min with uridine-3h and fractinated as described in Materials and Methds. The nuclelar RNA labeled in a 10-min pulse cnsists f 45S RNA and a hetergeneus species f lwer mlecular weight crrespnding, apparently, t partially synthesized chains (19). Sme f the decrease in rate f labeling f 45S RNA culd be due t an alteratin in the equilibratin f nucletide pls with the exgenus label. Althugh synthesis f all species f RNA declines under these cnditins f prtein synthesis inhibitin, the decrease is much less fr bth the nucleplasmic and cytplasmic species f RNA. Unless the nucletide pl supplying the ribsmal precursr is distinct frm that supplying the ther cellular species, it must be cncluded J O F- 0 that the rate f ribsmal precursr RNA synthesis is rapidly diminished when prtein synthesis is inhibited with cyclheximide. N evidence f separate nucletide pls has yet been bserved fr mammalian cells. One additinal cnclusin can be drawn frm the gel electrphretgrams shwn in Fig. 2b. A diminished rate f synthesis can be due t either fewer plymerase mlecules functining r t a decreased rate f transcriptin by a cnstant number f plymerase mlecules. If decreased transcriptin rate were respnsible fr the inhibitin f precursr frmatin, the rati f partially synthesized mlecules t cmpleted 45S mlecules wuld be increased. N such increase is apparent in Fig. 2b, and it is cncluded that the THE JOURNAL OF CELL. BIOLOGY VOLUME 41, 1969

5 time t cmplete a 45S mlecule remains cnstant and that fewer plymerase sites are functining in the inhibited cells. If synthesis f 45S RNA precursr is inhibited with actinmycin, the amunt f 45S RNA in the nuclelus decreases (11). Within 20 min after additin f actinmycin, 45S RNA has nearly disappeared frm the nuclelus (2). It might be expected that a decline in the rate f synthesis f 45S RNA during prtein synthesis inhibitin wuld be accmpanied by a decrease in the amunt f this mlecule in the nuclelus. Hwever, as shwn in Fig. 3, the decline in the rate f synthesis in the presence f cyclheximide is nt accmpanied by a decrease in the net amunt f 45S RNA. 45S RNA cntent f nucleli is measured tw different ways : First, the ptical density f the 45S peak in an acrylamide gel is measured and the area assciated with this peak is determined. Alternatively, cells are allwed t incrprate uridine-' 4 C fr 1 hr befre the administratin f cyclheximide. This length f time is sufficient t saturate the 45S species as previusly shwn (2). Thus, the radiactivity becmes equivalent t the ptical density and the amunt f RNA labeled in 45S is measured after administering cyclheximide. The results f such an experiment are dependent upn reprducible recveries between samples. Recveries are checked with a small aliqut f uridine- 3H-labeled cells, which is added t the drugtreated cells befre fractinatin. The recvery f tritium-labeled 45S RNA shuld be cnstant fr each sample. The amunt f 3H-labeled RNA is measured and used t crrect fr small differences in recvery. The ptical density cntributed by the tritium-labeled RNA crrespnds t nly 2 % f the ptical density frm the drug-treated cells. Thus, measurements f the ptical density f 45S RNA are nt significantly affected. The results f measuring ptical density r radiactivity in 45S RNA agree perfectly. The RNA present in the nuclelus after inhibitin f prtein synthesis is shwn in Fig. 3. The amunt f the majr species changes very little. The amunt f radiactive 45S RNA present in prelabeled cells after the additin f cyclheximide is als pltted. The nuclelar cntent f 45S RNA falls very little during the I st hr f prtein synthesis inhibitin. In sme experiments the amunt f 45S actually rises fr 30 min befre declining slwly. 5 5 I I M 10 5 a MIN ACTINOMYCIN SLICE NO. FIGURE 4 4 X 107 cells were suspended in 20 ml f medium and labeled with uridine-5-3 H as in Fig. 2. After 10 min, ne half f the culture was pipetted nt crushed frzen Earle's saline. Actinmycin D at a cncentratin f 20 tag/ml was added t the remaining half and the cells were incubated fr 15 min mre. Tw cultures f 4 X 10 7 cells each were treated with cyclheximide fr 5 and 45 min, and then labeled fr 10 min and incubated with actinmycin as abve. Nucleli were prepared and RNA was extracted as described in Materials and Methds. RNA was analyzed n plyacrylamide gels as in Fig O- O-O 10-min label ; Actinmycin D, 15 min. Since the rate f synthesis f 45S RNA apparently decreases significantly and the amunt f 45S RNA decreases very little, it must be cncluded that the rate at which RNA leaves the 45S stage has been reduced. This reductin in the rate f prcessing is demnstrated directly in the experiments the results f which are shwn in Fig. 4. Cntrl cells are labeled with uridine- 3 H fr 10 min and a very high cncentratin f actinmycin (20 µg/ml) is added t the culture. WILLEMS ET AL. RNA Synthesis and Prcessing during Prtein Synthesis Inhibitin 18 1

6 TABLE I Reductin f Rate f RNA Prcessing during Prtein Synthesis Inhibitin 45S 32S 45S remaining after actinmycin 45S decay in 32S rpm Cntrl 32,304 12, rpm Cntrl + 15 min. actinmycin 4, min cyclheximide 16, min cyclheximide actinmycin 55 min cyclheximide 7, min 9,379 2, min cyclheximide + 15 min actinmycin Radiactivity in the 45S and 32S peaks in Fig. 4 was estimated. A backgrund was estimated frm the hetergeneus RNA migrating t the left f each peak. This backgrund was assumed cnstant thrugh the peak regin and subtracted. Other cultures are treated with cyclheximide fr varius lengths f time befre labeling and then treated identically. A sample is taken at the same time that actinmycin is added, and a secnd sample 15 min later. In the cntrl, the labeled 45S RNA has decayed almst cmpletely after 15 min in actinmycin. This agrees with previus measurements f the lifetime f 45S RNA. In cntrast, cells which were pretreated with cyclheximide have 55 0/,, f labeled RNA remaining as 45S after 15 min in actinmycin. The results are summarized in Table I. Anther imprtant pint is illustrated by this experiment. The amunt f radiactivity appearing in 32S RNA after the administratin f actinmycin withut cyclheximide is apprximately 40 j f the radiactivity which has disappeared frm 45S. This is in apprximate agreement with previus measurements, which indicate that the mlecular weights f 45S and 32S are in the rati f abut 2 :1 and that the percentage f uridine is the same in bth mlecules. The prcessing f 45S in the presence f actinmycin therefre appears relatively nrmal. In the presence f cyclheximide, althugh the rate f transfrmatin is slwer, the efficiency f transfrmatin is the same as in untreated cells. Thus, abut 407, f the radiactivity disappearing frm 45S appears in the 32S peak. 4,106 1, It shuld be nted that the accuracy f the experiment shwn in Fig. 4 is limited by several cnsideratins. First, the result is sensitive t differences in recvery, which alter the apparent amunt f 45S RNA that has decayed. In this experiment, the amunt f 32S RNA was used t crrect fr recvery differences, since this slwly prcessed RNA species changes very little during a 15-min incubatin in actinmycin. Anther pssible surce f errr is the cntinued synthesis f 45S RNA fr a shrt time after the administratin f actinmycin. Cntinued synthesis will lead t an underestimate f 45S RNA which has decayed. A high cncentratin f actinmycin (20 µg/ml) was used in these experiments. Apprximately the same results are btained with 5 ug/ml f actinmycin. It can be cncluded that cntinued synthesis f 45S RNA was prbably nt an imprtant surce f errr. The cnstant efficiency f transfrmatin applies nly t the frmatin f 32S RNA. The 18S RNA, which is cleaved frm the 45S precursr in cntrl cells and rapidly exprted t the cytplasm, is frmed in lw amunts after the administratin f cyclheximide. This is illustrated in the experiment shwn in Fig. 5. Cells were labeled fr 60 min with radiactive uridine and fractinated as described in Materials and Methds. The nuclelar and cytplasmic 182 THE JOURNAL OF CELL BIOLOGY VOLUME 41, 1969

7 CPM 1000 CONTROL I 5'ACTIDIONE + 60' LABEL 60 1 ACTIDIONE + 60' LABEL NUCLEOLI OD CYTOPLASM 3000 CPM 1000 I ~ - I f I FRACTION FIGuuE 5 2 X 10 7 cells were suspended in 10 ml f medium and labeled fr 60 min with diluted uridine- 2-14C as in Fig. 3. Tw cultures f 2 X 10 7 each were treated with cyclheximide fr 5 and 60 min, and then labeled as abve. Cytplasm and nucleli were prepared and the RNA was extracted as described in Materials and Methds. Nuclelar RNA was analyzed n plyacrylamide gels as in Fig. 2. Cytplasmic RNA was analyzed n SDS sucrse gradients as described in Materials and Methds. Gradients were spun at 25 C fr 16 hr at 24,000 rpm in the SW 25.3 rtr f the Beckman I-2 ultracentrifuge (Beckman Instruments, Inc., Fullertn, Calif.) and eluted thrugh the flw cell f the Gilfrd recrding spectrphtmeter. Fractins were cllected, precipitated with 10% trichlracetic acid, and radiactivity was assayed as in Fig Optical density (OD) ; - - Radiactivity. fractins were extracted and the RNA was analyzed n acrylamide gels fr the nuclelus and n sucrse gradients fr the cytplasm. Cnsiderable radiactivity appears in cytplasmic 18S RNA after 60 min. Previus results, in fact, indicate that the 18S and 32S RNA appear cncmitantly (11). In anther culture, treated fr 5 min with cyclheximide befre labeling, there is n radiactivity in cytplasmic 18S RNA after a 60-min labeling perid. Thus, 32S RNA is frmed withut the cncmitant appearance f 18S RNA. It will be shwn later that n 18S RNA is present, either in the nucleli r in the nucleplasms f these cells. Since there is cmpelling evidence that bth 18S and 32S RNA are cntained in a single 45S precursr (20-23), these experiments indicate that the 18S RNA is degraded subsequent t the transfrmatin f 45S t the lwer mlecular weight prducts. A OD similar result has been btained by ther wrkers (6-8). It is interesting t nte that, despite the declining rate f synthesis f ribsmal precursr and the increased prcessing time required fr 45S RNA, the amunt f 32S RNA, as measured by the ptical density in this peak, in Fig. 5, remains cnstant. This suggests that the nuclelus maintains an rdered prcessing, althugh at a reduced rate, s that the same number f newly synthesized mlecules enter the 32S species as leave, despite the inhibitin f new prtein synthesis. It is pssible that the regulatin is due t the maintenance by the nuclelar structure f a cnstant level f 32S RNA, and that rates f synthesis and prcessing adjust themselves t keep the amunt f 32S RNA relatively cnstant. The nucleli f cells treated fr lng perids f time with high levels f cyclheximide became WILLEMS ET AL. RNA Synthesis and Prcessing during Prtein Synthesis Inhibitin 1 83

8 CPM NUCLEOLUS NUCLEOPLASM CYTOPLASM CPM CPM 54' g, SEDIMENTATION CONSTANT (S) FIGURE 6 2 X 108 cells were suspended in 80 ml f medium and incubated fr 30 min with cyclheximide (600 ug/ml) and then labeled with uridine-2-14c as in Fig. 3. Samples f 5 X 10 7 cells were taken 54, 90, 120, and 240 min later. Cells were fractinated int nucleli, nucleplasm, and cytplasm, and the RNA was extracted as described in Materials and Methds. RNA was analyzed n sucrse density gradients in SDS sucrse buffers as described in Materials and Methds, eluted as in Fig. 5, and the radiactivity was assayed as in Fig. 2. Centrifugatin was at 25 C fr 16 hr at 21,000 rpm fr nuclelar RNA and 16 hr at 24,000 fr nucleplasmic and cytplasmic RNA in the SW 25.3 rtr f the Beckman L-2 ultracentrifuge. P 120' 240' aberrant in functin with greatly reduced levels mature 28S RNA, which eventually appears in f RNA synthesis and rates f prcessing. Never- the cytplasm. In the experiment shwn in Fig. 6, theless, even after prlnged perids f prtein HeLa cells were treated with cyclheximide fr synthesis inhibitin, the nuclelus is able t frm 1 hr. The greatly reduced rate f prcessing cm- 184 THE JOURNAL OF CELL BIOLOGY. VOLUME 41, 1969

9 100 NUCLEOPLASM NUCLEOLUS TIME (MIN) AFTER REVERSAL FIGURE cells were suspended in 50 ml f medium and divided as fllws : 2 X 10 7 cells were labeled fr 10 min with uridine-5-3h as in Fig X 107 cells were incubated with cyclheximide fr 60 min. At this time 2 X 10 7 cells were remved and labeled as abve. The remaining cells were washed three times in cld medium, resuspended in warm medium, incubated fr 5, 30, and 60 min, and then labeled as abve. Nucleli were prepared and RNA was extracted as described in Materials and Methds. Nuclelar RNA was analyzed n plyacrylamide gels and the ttal radiactivity in 45S RNA was measured as in Fig. 2. The nucleplasmic fractin was reserved and an aliqut was measured as in Fig. 2, except that an additinal sample was made 0.3 N NaOH and incubated vernight at 37 C. Acidprecipitable radiactivity remaining after this hydrlysis was a measure f incrpratin f label int DNA and the samples were crrected by this amunt t give ttal acid-precipitable radiactivity specifically in RNA. pared t the rate in nrmal cells can be seen. Sme 32S RNA is present after 1 hr f labeling, but an additinal 3 hr f incubatin is required befre a significant amunt f 28S RNA emerges int the cytplasm. This experiment is perfrmed with 600 jug/ml f cyclheximide. At this cncentratin the rate f prtein synthesis is less than 1 % f cntrl. It is pssible that the meager prductin f 28S RNA is due t the residual prtein synthesis. Hwever, the amunt f 28S RNA prduced is in excess f 1 % f cntrl cells, and the results suggest that a pl f structural prteins exists as previusly reprted (6). These experiments were perfrmed by tising sucrse gradients fr analyzing all fractins. It is apparent that n 18S RNA is prduced under these cnditins, which include a lng preincubatin in cyclheximide. One further experiment indicates that the decline in nuclelar RNA synthesis is nt due t irreversible damage f cellular structure resulting frm the presence f the prtein synthesis inhibitr. Cells were expsed t cyclheximide fr 1 hr and the rate f 45S RNA synthesis was measured. The drug was remved and the recvery f 45S RNA synthesizing capacity was fllwed. The results are presented graphically in Fig. 7. The rate f 45S synthesis declined t 18% f the value in cntrl cells while the drug was present. After the remval f the drug, synthesis returned t 45% f cntrl within 10 min. The recvery cntinued, althugh mre slwly, and reached 60% f the cntrl after 60 min. The rapid, althugh partial, recvery suggests that much f the RNA synthesizing machinery remains intact and is ready t functin as sn as new structural prteins becme available. DISCUSSION The results presented in this reprt shw that the nuclelus f HeLa cells respnds prmptly t cessatin f prtein synthesis. Ribsmal RNA synthesis slws drastically. Hwever, the prcessing time f the 45S RNA precursr als increases s that the RNA cntent f the nuclelus remains relatively cnstant. In nrmal cells the 45S RNA precursr t ribsmal RNA has a lifetime f abut 15 min, after which 18S RNA and a guansine-cytsine-rich species f RNA are cleaved and remved frm the nuclelus, leaving 32S RNA (10, 15, 16). Under cnditins f prtein synthesis inhibitin, the lifetime f 45S RNA is increased and, althugh 32S RNA is frmed frm cleavage f the precursr mlecule, n 18S RNA appears. Hwever, the prductin f 32S RNA appears nrmal in the sense that ne mlecule f 32S is prduced fr every mlecule f 45S that is cleaved. This is cncluded frm the results f the decay f 45S RNA in actinmycin. Apprximately 40% f the radiactivity which disappears frm 45S appears in 32S in bth inhibited and cntrl cells. This fractin f radiactivity is predicted n the basis f the mlecular weights f the varius species. The increase in prcessing time measured by actinmycin decay f 45S RNA is cnsistent with the decrease in the rate f synthesis f 45S RNA withut a cncmitant decline in the amunt f 45S RNA. This increase in prcessing time has appeared by 15 min after the inhibitin f pr- 1AWILLEMS ET AL. RNA Synthesis and Prcessing during Prtein Synthesis Inhibitin 1 8 5

10 tein synthesis. Hwever, the increase in prcessing time des rit cntinue t cmpensate fr the decreased rate f synthesis, and the amunt f 45S RNA in the nuclelus begins t decline at abut 1 hr. The nrmal frmatin f 32S RNA frm its 45S precursr in cyclheximide-treated cell'i cntrasts with results btained frm ther methds f interfering with nuclelar functin. Results similar t thse described here are btained with purmycin (7). Hwever, the 32S RNA frmed is nt prcessed t 28S RNA. This is shwn t be a result f the prematurely terminated plypeptides prduced by purmycin. During plivirus infectin, 18S RNA is frmed nrmally but 32S RNA frmatin is greatly suppressed (15, 25). In this latter case there is a buildup f the 41S intermediate, suggesting that the times f prcessing in nrmally shrt-lived states have been greatly increased. The drug crdycepin prduces prematurely terminated 45S mlecules. Under these cnditins sme 18S RNA is frmed but 32S RNA is suppressed (26). The functin f the nuclelus is clearly regulated in part by the availability f a prtein cmpnent, which is pssibly, but nt necessarily, a ribsme structural cmpnent. It is pssible t speculate abut the nature f the signal that regulates synthesis and prcessing time. One pssibility, which is in agreement with results shwn here, is that the amunt f 32S RNA physically ccupying the nuclelus determines the rate f synthesis and prcessing f 45S RNA. At least the nuclelus functins s as t keep the amunt f 32S cnstant as shwn in Fig. 5. Als, the rati f 45S t 32S labeled during a lng incrpratin becmes cnstant as shwn by the results in Fig. 6. The rati f radiactivity in 32S cmpared t 45S in the cells labeled after a lng perid f prtein synthesis inhibitin appraches the rati fund in nrmal cells. The time necessary t apprach this rati is f curse much lnger than in nrmal cells. Althugh under cnditins f inhibitin the nuclelus functins s as t frm the 32S successfully and maintain a relatively cnstant level, the successful frmatin f 18S RNA frm the 45S precursrs ceases sn after the inhibitin f prtein synthesis. Tw mdels are pssible t explain the regulatin f 45S RNA synthesis. The results shwn in Fig. 2 suggest that decreased synthesis is due t fewer functining plymerase mlecules. Thus, either nrmally unstable plymerase mlecules break dwn and are nt replaced under cnditins f prtein synthesis inhibitin, r plymerase mlecules are immbilized and begin t functin nly when prtein synthesis inhibitin is reversed. The experiment shwn in Fig. 7 indicates rapid recvery in the rate f RNA synthesis after the remval f cyclheximide. This rapid, althugh partial, recvery suggests that the plymerase mlecules exist and can cmmence functin as a new supply f prtein fr nuclelar prcessing becmes available. Hwever, experiments f this type cannt determine which f the tw mdels f RNA synthesis regulatin is crrect. It may seem dd that nuclelar functin is inhibited while a pl f prteins sufficient t finish ribsmes still exists. It appears that the highly rganized structure f the nuclelus functins slwly when less than a nrmal supply f prtein is available. This research was supprted by The Natinal Institute f Health, Grant N. CA and by the Natinal Science Fundatin, Grant N (GB). Sheldn Penman is a recipient f a Career Develpment Award N. GM frm the Natinal Institute f Health. We gratefully acknwledge the excellent technical assistance f Irene Furnier. Received fr publicatin 16 September 1968, and in revised frm 27 Nvember REFERENCES 1. PERRY, R. P The cellular sites f synthesis Base cmpsitins f fractins f nuclear f ribsmal and 4S RNA. Prc. Nat. Acad. and nuclelar ribnucleic acid btained by Sci. U.S.A. 48 :2179. sedimentatin and chrmatgraphy. J. Bil. 2. PENMAN, S., 1. SMITH, and E. HOLTZMAN Chem. 241 :1544. RNA metablism in a particulate site in the 4. HAY, E., and J. REVEL The fine stru-ture HeLa cell nucleus. Science. 154 :789. f the DNP cmpnent f the nucleus. J. Cell 3. MURAMATSU, M., J. HODNETT, and H. BUSCH. Bil. 16 : THE JOURNAL OF CELL BIOLOGY. VOLUME 41, 1969

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