Mechanism of turnover of rapidly labeled RNA in rat liver nuclei

MECHANISM RAPIDLY

OF LABELED

LIVER

TURNOVER RNA

OF IN

RAT

NUCLEI*

ROBERT B. HURLBERT, DOROTHY HARDY, MARIAN STANKOVICH and ROBERT D. COLEMAN Department of Biochemistry, The University of Texas, M. D. Anderson Hospital and Tumor Institute, at Houston, Texas INTRODUCTION WHEN R N A is labeled for brief periods in living cells, almost all of the label is found in nuclear RNA, much of it in the 45 S preribosomal R N A of the nucleolus and a usually larger proportion is found in the extranucleolar RNA. Following the time course of events after a pulse-labeling of the nucleolar R N A has led to the concept that the 45 S preribosomal R N A is degradatively processed to the 18 and 28 S R N A molecules of the cytoplasmic ribosomes; about one-half of the originally transcribed molecule is thereby lost in stepwise fashion (see ref. 1). Similar attempts by kinetic studies to follow the fate of the extranucleolar R N A accounts for only a small fraction of the original pulse label as cytoplasmic polysomal messenger RNA, yet the labeled R N A is soon lost from the nucleus. The rapidly labeled extranucleolar R N A has a high proportion of adenine and uracil, resembling the ( C + G ) / ( A + T ) composition of DNA, and on sucrose gradients is distributed heterogeneously over a range of 10-60 S. This type of RNA, either labeled or unlabeled, does not accumulate in the nucleus; most of the nuclear as well as the cytoplasmic RNA is of the ribosomal high G + C type (I am here omitting discussion of the various transfer and 4-6 S RNAs). Thus, it appears that this type of R N A - - u p to 80 or 90 ~o of it--is rapidly degraded, presumably in the nucleus. Harris (2) was probably the first to point out the anomalous nature of R N A which is rapidly synthesized and then almost as rapidly degraded without leaving the nucleus. Possibly because he interpreted the data as a challenge to the developing view that a labile messenger R N A is synthesized in the nucleus and transported to the cytoplasm, the observation was not widely recognized. More recent * A b b r e v i a t i o n s . The following abbreviations are used: C, G, A, T and U refer to the cytosine, guanine, adenine, thymine and uracil bases or nucleotides of DNA or RNA. PCA is perchloric acid, Tris is tris(hydroxymethyl)aminomethane. 323

324

ROBERT B. HURLBERT et

al.

documentation of the above observations and characterization of this rapidly labeled, heterogeneous, giant, extranucleolar, DNA-like, labile form of RNA, which contains some sequences in common with polysomal messenger RNA, have been provided by Watts (3), Shearer and McCarthy (4), Soeiro et aL (5), Soeiro and Darnell (6) and others who are cited in these papers. The most recent and comprehensive concept is that of Georgiev and his group (7) who provide evidence that a large transcriptional unit is rapidly synthesized, then degradatively processed from the 5'-end so that the remaining 3'-end is precursor of polysomal messenger RNA. If messenger RNA is thus derived as a small fraction of a large part of RNA, recently transcribed from DNA, it would seem likely that control mechanisms are needed to regulate the selection of molecules, or portions thereof, to be translated. Indeed, such a mechanism would be theoretically beneficial to augment the transcriptional step in control of genetic expression, provided that the rescue of specific RNA molecules or messages is coupled in some way to metabolic control elements such as hormones or metabolic effectors. Thus, RNA representing redundant gene copies, nongene transcriptional sequences, and metabolically unnecessary messages could be routinely shredded up while messages for enzymes or other protein factors, needed for phenotypic expression or correction of momentary imbalances in metabolism, would be rescued. The very existence of such an apparently wasteful degradation of large amounts of RNA (in conjunction with some faith in the Wisdom of Nature) suggests that the potential regulatory benefits of the degradation must outweigh the disadvantages of metabolic inefficiency. As a first step in study of this problem, we examined the mechanism of the degradation of rapidly-labeled RNA, using pre-labeled isolated nuclei as experimental material. Our rationale was that the degradation might prove to be a relatively facile and efficient turnover between the RNA and nucleoside-5'-triphosphate pools, rather than a much more energy-demanding and irreversible degradation of the RNA to 2'(3')-nucleotides, nucleosides and free bases. Justification for this rationale was provided by our early observation that the specific activity in vivo of RNA uracil (from citric-acid washed nuclei) reached about one-half the value and paralleled the specific activity of cellular uridine nucleotides (8), plus the finding of Harris (9) that some labeled 5'-adenosine nucleotides are released from adenine-labeled nuclei during incubation, plus the description by Lazarus and Sporn (10) of a nuclear exonuclease producing nucleoside-5'-phosphates from RNA. The basic plan in these experiments has been to inject 14C-orotic acid, 3H-adenine or 32p.phosphat e into rats, isolate RNA-labeled nuclei from the liver after ½ to 2 hr, and incubate the nuclei in unlabeled media previously shown to be capable of supporting RNA synthesis (11, 12), followed by determination of label in RNA and in cold PCA-soluble nucleotides. In this series of experiments we did not attempt to discriminate between degradation

TURNOVER OF RAPIDLY-LABELED NUCLEAR

RNA

325

of the rapidly labeled extranucleolar R N A and degradation of the also rapidly labeled nucleolar preribosomal RNA. MATERIALS

AND METHODS

Materials. Unlabeled nucleotides and other chemicals were obtained from commercial sources. The rats used were 140-160 g Sprague-Dawley females.

Preparation of nuclei. All sucrose media contained 3 mM MgCI2 and 0.05 M glycine, finally adjusted to p H 7.4 with KOH. "Isotonic sucrose" solutions contained 0.25 M sucrose. All preparations were maintained at 0-4 °. Rats were fasted overnight, killed and bled, and the livers were rapidly perfused with cold isotonic sucrose. The livers were minced and strained with a Harvard Tissue Press and the pulp was quickly weighed, then suspended in 4 ml of isotonic sucrose per g of pulp with a loose-fitting Dounce homogenizer. The suspension was diluted with 4 ml/g more and homogenized with a tight fitting Dounce homogenizer, about four strokes, or as needed to disrupt the majority of the cells. The homogenate was diluted with a total of 19 ml of isotonic sucrose per g (5 ~o suspension) and centrifuged at 400 rpm in a horizontal rotor to remove clumps and cells. The supernatant (carefully removed) was centrifuged at 1500 rpm (500 x g, horizontal rotor) to sediment nuclei. The nuclear pellet was rehomogenized briefly in one-half the homogenate vol of isotonic sucrose, distributed in centrifuge tubes, and an equal vol of 0.5 M sucrose solution was layered underneath by use of a syringe and a long needle. After centrifugation at 1500 rpm, the supernatant was discarded and the nuclei were resuspended in one-half the homogenate vol of isotonic sucrose. The 400 rpm spin to remove clumps, the underlayering and the 1500 rpm spin were repeated. Finally, the nuclei were packed at 2000 rpm, volume was estimated, and they were suspended in 4 times this vol of isotonic sucrose. The R N A / D N A ratio was 0.5-0.7, clumping and visible contamination by cytoplasmic debris was minimal, and the ratio of nuclei to whole ceils was 15 or more. These are minimally-altered, rather than "clean", nuclei. Because our objective was to simulate the response of nuclei in vivo, we did not wish to risk removal of nuclear constituents by more rigorous and even less "physiological" purification. Preparation of"S3fraction". Rat livers were perfused and minced as above, but homogenized and centrifuged at 2000 rpm as a 20~o homogenate in isotonic sucrose. The supernatant was centrifuged at 200,000 x g in a fixed angle rotor for 1 hr. The supernatant ("$3") was dialyzed in a cellulose membrane against 70 vol of 0.25 M sucrose-3 mM MgCI2-O.02 M Tris-Cl (pH 7.4) for 1 hr followed by 70 vol for 2 hr at 4 °. The preparation was quick-frozen and stored at - 2 0 ° in 2-ml portions.

326

ROBERTB. HURLBERTet al.

Incubation of nuclei and analysis of RNA degradation. The standard medium for incubation of the nuclei contained 250 mM sucrose, 100 mM Tris-C1 (pH 8.5), 5 mM MgC12, 5 mM phosphoenolpyruvate, 0.5 mM each of ATP, UTP, CTPand GTP, 20 pg/ml of pyruvate kinase (E.C. 2.7.1.40, Sigma Chemical Co.) and nuclei containing 0.3-0.5 mg of DNA, in a total vol of 2.0 ml. In specified eases 0.20 ml of dialyzed "$3" fraction was included. Incubation was at 38 °, in 12-ml centrifuge tubes, with shaking and occasional stirring. The reaction was stopped by chilling and addition of 2.0 ml of cold 0.8 M PCA, followed by centrifugation. The supernatant was kept separately and the precipitate was washed 3 times with 8 ml of cold 0.4 N PCA (washings discarded). The precipitate was then extracted with 4.0 ml of 0.4 N PCA at 100° for 10 min. The "cold PCA" and "hot PCA" extracts were each adjusted to the same vol (usually 4.1 ml) in graduated hematocrit tubes and two 0.5-ml aliquots were removed for determination of radioactivity. To the remaining cold PCA extract was added carrier nucleotides (1-2 pmoles each), the solution was adjusted to the pH 5-6 with K O H and chilled further. The KC104 was removed by centrifugation and the solution was concentrated 5-fold in a vacuum desiccator over N a O H and CaC12 in the cold. The hot PCA extract was also analyzed for RNA and D N A by the orcinol and diphenylamine procedures (11).

Separation of labeled nucleotides. The concentrated cold PCA extracts were spotted or streaked on 40 cm long Whatman 3 M M sheets, about 5 pl per cm. The papers were moistened, provided with wicks, and sandwiched between plastic films. The buffer was 0.025 ~ citrate (citric acid adjusted to p H 5.5 with NaOH). Conditions were 2000-2500 V and 1.5-2 mA/cm width at about 4 ° for 11 hr in a Savant flat-bed apparatus. The members of each nucleotide series (e.g. uridine, 5'-UMP, UDP, UTP) were discretely separated, in that order (uridine at the origin, UTP about three-quarters way towards the positive electrode). CTP and inorganic phosphate ran almost with UTP. CDP, ATP and G T P ran in a cluster with UDP. ADP and G D P ran together. In the 32p.experiment s cited, the components of these clusters were separated with modest success by electrophoresing them from the cut strips of 3 M M paper onto DEAE paper in 0.10 M citrate buffer (pH 3.35) at right angles to the first run. The papers were dried, the spots visualized under a UV lamp and cut out. With 14C-labeled material the spots were in some cases counted directly in toluene-based scintillation fluid. In other cases, and with 3H-label, they were eluted with water by centrifugation in a pierced plastic tube resting inside a tapered centrifuge tube; the eluate was concentrated or adjusted to 0.5 ml for scintillation counting. Other areas of the electropherogram, as well as a duplicate sample spot, were similarly counted for quantitation.

TURNOVER OF RAPIDLY-LABELED NUCLEAR R N A

327

Anion-exchange chromatography was conducted essentially as previously described (13) with a 12 x 1 cm column of Dowex-l-formate, linear gradient, 200 ml H20 and 200 ml of 4M formic acid in the mixer and reservoir, followed by 200 ml of 4 rd formic acid and 200 ml of 4 u formic acid-l.2 t,l ammonium formate in the second gradient.

Use and measurement of radioactivity. [6-14C]-orotic acid (Volk Radiochemical Co., 3 mCi/mmole) or [G-3H]-adenine (New England Nuclear Co., 10 Ci/mmole), or [32p]_sodium phosphate (Hastings Radiochemical Co., carrier-free), in 1-2 ml of isotonic NaC1, were injected intraperitoneally into the rats ½ to 2 hr prior to sacrifice. The standard method of counting was to prepare the sample in 0.5 ml of 0.4 MPCA and to mix this with 10.0 ml of Aquasol (New England Nuclear Corp.). Where deviations from this mixture were necessary, corrections for efficiency were determined and made. Standard efficiencies were 78~o for 14C and 22~o for 3 H in a NuclearChicago Mark I instrument. RESULTS

Characteristics of the Nuclear RN,4-degradation System Figure 1 and Table 1 illustrate several experimental observations which will be described at intervals in the following text. To begin with, they are representative of the fate of x4C-orotic acid-labeled RNA during incubation of isolated nuclei, as observed in a number of experiments. As detailed in Methods and the legends, the labeled nuclei were prepared from rat liver in isotonic sucrose and were incubated in a medium which preserved morphology well and permitted incorporation of nucleoside triphosphates into RNA. In Fig. 1 the "suc-Mg" solid-line curve illustrates that routinely some 40-60 of the cold PCA-pr¢cipitable uracil-labeled RNA was lost, most of this in the first 15-20 min. This label was recovered in the "PCA-soluble" fraction, as recorded in Table 1. In these experiments, we did not examine the nature of the residual RNA, but from previous experience (11, 12) expect it to be of the high G-C ribosomal type. The loss of unlabeled RNA was routinely one-half or less that of the labeled RNA (as illustrated in Table 1) indicating selectivity for the newly-labeled RNA. When 0.4 rd ammonium sulfate was included in the standard reaction mixture, the loss of both labeled and unlabeled RNA was almost completely prevented (data not shown, although the "AmSO4Mn ÷ +" solid line of Fig. 1 is comparable). In experiments of similar design, Watts (14) has published observations similar to the foregoing. In Fig. 1 we also show that RNA degradation and RNA synthesis can occur simultaneously. This is a single experiment in which the uracil-labeled nuclei were incubated in medium containing 3H-CTP to measure RNA synthesis;

328

R O B E R T B. H U R L B E R T e t

al.

SIMULTANEOUS SYNTHESIS AND DEGRADATION OF R N A

100

o_

o AmS°'-Mn'o I I

80

I !

< z

tr"

I..i~

60

< z

AmSO4-Mn++

er

-10 40

//"

"r

~ u c . - M g + +

6

r, -6

/

~6-

0

o

/ suc.-Mg ++

//

i

0

/

/I

I 10

•

-II-. . . . . . .

-I

I 20

I0

I 40

Minutes

FIG. 1. Simultaneous synthesis and degradation of RNA in rat liver nuclei in vitro. One rat received 3.3 gCi of 14C-orotic acid 2 hr before sacrifice and preparation of liver nuclei. The labeled nuclei were incubated in "sucrose-Mg ++'' or "AmSO4Mn + +" media containing ~H-CTP for the times indicated, then precipitated and analyzed as described in Methods, except that the cold PCA-soluble fraction was not retained and double-label scintillation counting was used. The solid lines represent loss of C14-uracil from the prelabeled RNA and the dashed lines represent incorporation of aH-CMP into newly synthesized RNA. The "sucrose-Mg + +" medium contained the same components as the standard incubation described in Methods except that 7.5 mM MgC12 and 1.0 mM of each nucleotide was used. The "AmSO4Mn ÷+'' medium differed by containing only 2.5 mM MgCI2 plus 1.2 naM MnC12 and 400mM ammonium sulfate. Each tube contained 20x 106 dpm of [5-aH] CTP. The zero-time nuclei contained 35,000 dpm per mg of nuclear DNA.

o n e portion o f nuclei was in the standard m e d i u m and the other portion in m e d i u m w h i c h included 0.4 M a m m o n i u m sulfate and substituted M n + + for M g ++ ions. W e (12) and others have previously shown that the "sucroseM g + +" m e d i u m permits synthesis o f m a i n l y high G - C nucleolar R N A , while the a m m o n i u m s u l f a t e - M n ÷ + m e d i u m greatly stimulates synthesis o f a high A - U - c o n t a i n i n g extranucleolar R N A ; these relative rates o f incorporation are s h o w n here. T h e point we wish to m a k e is that the degradation o f the nuclear R N A proceeded despite demonstrated synthesis in the isotonic s u c r o s e - M g ÷ + m e d i u m , and the synthesis o f high A - U R N A in the a m m o n -

t ?

15 15 15

d-"S3"

36.0 33.8 37.0

3.3 41.8 41.8 44.9 27.2

(%)

Percentage of nuclear C ~4 found as PCA-soluble

5 10 9

38 36

(%)

UR

4 3 3

31 36

(%)

UMP

70 70 63

5 1

(%)

UDP+ UTP

Percentage of PCA-soluble C ~4 as

R N A TO COLD

One rat received 3.35/tCi of 14C-orotic acid 2 hr before sacrifice. Liver nuclei were prepared and incubated as described in Methods, with the above additions. Loss of unlabeled R N A was measured as decrease of R N A / D N A ratio. Percent of total nuclear 14C recovered as cold PCA-soluble *4C represents degradation o f uracil-labeled RNA. The nuclei at zero time contained about 18,000 dpm per mg of DNA. Estimates of distribution of label in uridine compounds were made by electrophoresis at pH. 3.35, which was less definitive than the procedure reported in Methods. • Sample lost. t In other experiments, presence o f " S a" fraction diminished degradation of unlabeled R N A by about 50 ~ .

+ $3 +2'(3')-UMP, 10 mM +$3 + U R , 10 mM

15.4 13.6 13.6 4.9

(%)

0 15 15 15 15

Incubation time (min)

None None + Aetinomycin D, 10/tg + Phosphate, 30 mM + 2'(3')-UMP, 10 mM

Addition to standard incubation

Percentage degradation of unlabeled RNA

TABLE 1. EFFECTS OF VARIOUS INCUBATION MEDIA ON DEGRADATION OF URACIL-LABELED NUCLEAR P C A - s O L U B L E PRODUCTS

z

tt-n

O

t-n

~7 ~Z

> m

O

Z O

330

ROBERT B. HURLBERTet al.

ium sulfate-Mn ÷ ÷ medium proceeded despite a great reduction in degradation. The latter observation may be of note to those interested in the "ammonium sulfate effect" and its significance in interpretation of hormally activated changes in nuclear RNA-synthetic activities (see refs. 12, 15, 16). We suggest here that in the ammonium sulfate medium the synthesis of "heterogeneous nuclear" or "rapidly labeled DNA-like" R N A is continued and that this R N A is not degraded, whereas in the standard isotonic sucrose media this type of R N A is synthesized but immediately destroyed, leaving primarily the longer-lived nucleolar preribosomal RNA. In several experiments, the nucleotides CTP, G T P and U T P were omitted from the reaction mixture (data not shown). No significant effect on the degradation of the uracil-labeled R N A was noted. We have previously shown that omission of these nucleotides greatly diminishes the amount of incorporation of a single-labeled nucleotide (12). Inhibition of R N A synthesis by actinomycin D (Table 1) also had no effect on the degradation. It appears, therefore, that the degradation process is not correlated with or dependent on synthesis of new RNA, at least as far as these time periods are concerned. Nor is the synthesis dependent on prior degradation of RNA.

Identification of coM PCA-soluble products of degradation of uracil-labeled RNA. The nature of the radioactivity present in the cold PCA-soluble extracts was determined by electrophoresis after the extracts were neutralized and concentrated (details are given in Methods). The first part of Table 2 shows the results, under "Standard incubation". Orotic acid-labeled nuclei in the cold incubation mixture, extracted before beginning of incubation at 38 °, contained about I0 ~ of the label as acid-soluble material. Of this, about 58 was uridine monophosphate and 14 ~o was uridine. Upon incubation, more acid-soluble material was liberated, up to 65 ~o of the R N A at 15 min. At this time, 57 ~o of the acid-soluble label was recovered as uridine, 20 Yo as uridine monophosphate. In this experiment it was not attempted to determine whether the uridine monophosphate was the 2'-(3') nucleotide or the 5'-nucleotide, nor to identify conclusively the radioactivity in t h e " U D P " and " U T P " regions which did not migrate clearly with the carrier nucleotides and quite possibly included small oligonucleotides, nucleoside diphosphates, etc. In order to examine the proportions of 2'(3')-UMP and 5'-UMP products in separate but similar experiments, the cold PCA extract (adjusted to 1.0 M PCA) was heated at 100° for 30 min to degrade any oligonucleotides to products including uridine 2'(3')-phosphate and to degrade U D P and U T P to uridine-5'-phosphate. The neutralized hydrolysate was subjected to electrophoresis both before and after treatment with snake venom 5'-nucleotidase to selectively hydrolyze the 5'-UMP to uridine. The results indicated less than 3 0 ~ of the labeled nucleotides were 5'-UMP derivatives and 6 0 ~ were

331

TURNOVER OF RAPIDLY-LABELEDNUCLEAR R N A

2'(Y)-UMP derivatives. In other words, under the conditions thus far described for incubation of the nuclei, most of the degradation of R N A resulted in uridine, uridine-2'(Y)-phosphate and oligonucleotides. This result did not preclude the possibility that the uridine was derived by rapid dephosphorylation of uridine-5'-phosphate formed in significant quantities. One way of trapping any possible 5 ' - U M P was to provide conditions for vigorous phosphorylation to U D P and UTP. Nucleoside triphosphates, ATP, phosphoenolpyruvate and phosphoenolpyruvate kinase were already present in the incubation mixture; it seemed possible that the isolated nucleoli were deficient in suitable phosphokinases.

TABLE 2.

PRODUCTS OF DEGRADATION OF URACIL-LABELED NUCLEAR

Incubation conditions and time (min)

RNA

Percentage of cold coldPercentaget14Csolubleas°f °talpCA

~

PCA-soluble~UDP C~4 as UTP (%)

(%)

(%)

(%)

(%)

Standardincubation 0 5 15

10 38 65

14 40 57

58 40 20

16 8 10

10

Plus "Ss extract" 0 5 15

9 30 49

16 14 24

64 12 6

I0 8 8

10 66 61

Plus 0.04 MNaF 0 5 15

10 26 49

16 19 13

61 36 34

10 25 30

13 13 14

12 8

One rat received 16/to of [C14]-orotic acid for ½hr. Nuclei were prepared, incubated under standard conditions, extracted, and the cold PCA-soluble nucleotides were analyzed by eleetrophoresis as described in Methods. The content of C 14 in nuclear RNA at zero time was about 60,000 cpm per mg of nuclear DNA. UR is uridine; UMP is uridine monophosphate.

Nucleotide kinase activity of nuclei and "$3 extract". Table 3 shows a test of the ability of the isolated rat liver nuclei to phosphorylate labeled U M P and U D P . The nuclei (unlabeled) were incubated with the uridine nucleotides under the standard incubation conditions, except that as part of the experiment a rat liver fraction designated "$3" was included. This was the supernatant of an ultracentrifuged homogenate of rat liver in isotonic sucrose medium, which is known to contain active phosphokinases (17). Similar

332

ROBERT B. HURLBERT e t

al.

extracts o f rat liver are also k n o w n to c o n t a i n inhibitors o f r i b o n u c l e a s e (18), as will be discussed later. It is a p p a r e n t f r o m T a b l e 3 t h a t the nuclei by themselves r a p i d l y d e p h o s p h o r y l a t e d 3 H - U M P to uridine with no c o n v e r s i o n to U D P a n d U T P , a l t h o u g h they were able to convert 1 4 C - U D P to U T P quite well for 10 min. T h e a d d i t i o n o f the " $ 3 " extract b r o u g h t a b o u t p h o s p h o r y l a t i o n o f 77 ~ o f the 3 H - U M P to U D P a n d U T P with loss o f only 19 ~ to uridine, a n d i m p r o v e d even m o r e the p h o s p h o r y l a t i o n o f U D P .

TABLE 3. A S S A Y FOR NUCLEOTIDE KINASE ACTIVITIES OF NUCLEI AND " $ 3 " EXTRACT

Incubation conditions and time (min)

Percent of label recovered as UDP

UTP

UR

UMP

3H-UMP, nuclei only 0 10 20

2 78 88

98 20 11

3H-UMP, nuclei + "S 3" 10 20

19 34

3

8

11

20

69 32

14C-UDP, nuclei only 0 10 20

1

4

16 32

7 7

69 7 10

26 70 51

14C-UDP, nuclei + "S a" 10 20

7 19

2 16

8

28

83 35

(%)

(%)

(%)

%

Unlabeled rat liver nuclei were prepared and incubated by the standard procedures except that CTP and GTP were omitted and either 2/tmoles of [G-3H]-UMP (2/tCi, New England Nuclear Corp.) or 2gmoles of [U-14C]UDP (0.32ttCi, Amersham-Searle) were added in the incubation mixture. "$3" is a dialyzed extract of rat liver and the reaction products were analyzed by electrophoresis with 0.5 ttmole of each compound as carrier, as described in Methods.

Effect o f " S 3 " extract on recovery o f RNA-uracil as UTP. T h e s e c o n d p a r t o f T a b l e 2 shows the effect o f a d d i n g the " $ 3 " extract o f r a t liver d u r i n g i n c u b a t i o n o f orotic a c i d - p r e l a b e l e d nuclei. A g a i n at zero time, U M P p r e d o m i n a t e d a m o n g the acid-soluble p r o d u c t s . The rate o f R N A d e g r a d a t i o n in the presence o f " $ 3 " was diminished, consistently with o b s e r v a t i o n s in all similar experiments. T h e m o s t striking o b s e r v a t i o n was t h a t u n d e r these m o r e

TURNOVER OF RAPIDLY-LABELED NUCLEAR

RNA

333

complete phosphorylating conditions the predominant product was UTP. The labeled UTP spot was discrete, and was identified as such by various other chromatographic tests in the presence of carrier UTP. In similar experiments, up to 70 ~o conversion to UTP has been obtained. In still other experiments to determine the amount of 5'-UMP derivatives by acid hydrolysis, followed by treatment with 5'-nucleotidase, 80-90 ~o recovery of label as 5'-UMP nucleotides was obtained. Lazarus and Sporn (10, 19) reported that nuclei contain an exonuclease which hydrolyzes RNA stepwise to liberate 5'-nucleotides, and that this nuclease was inhibited by 2'(3')-nucleotides and NaF. Table 1 shows that 10 mM 2'(3')-UMP inhibits degradation of both labeled and unlabeled nuclear RNA, although the action does not appear to be on the exonuclease. The third part of Table 2 shows the effect of inclusion of 0.04 M NaF in the reaction mixture in the absence of "$3". The rate of RNA degradation was diminished. Again, UMP predominated at zero time. In this case, relatively little uridine was formed, and the label accumulated in the UMP and UDP regions. The radioactive spots did not migrate closely with carrier nucleotides, and, in other experiments using the acid hydrolysis and 5'-nucleotidase procedure, very little 5'-nucleotide was indicated to be present. When both 0.04 MNaF and "Sa" were added in an experiment similar to the one described in Table 2, the amount of degradation was much less: 5 ~o, 7 ~ , and 12 ~ at 0, 5 and 15 min, respectively. The combination of "$3" and NaF inhibits almost as greatly as 0.4 M ammonium sulfate. It seems apparent that the ribonuclease inhibitor known to be in the "$3" (18) inhibits a cytoplasmic endoribonuclease which forms 3'-phosphate-ended oligonucleotides and 2'(3')-UMP while at the same time permitting production and phosphorylation of 5'-UMP to UDP and UTP. The NaF then apparently inhibits an exonuclease similar to that described by Lazarus and Sporn, but permits the endonuclease to function. The NaF may also inhibit the phosphatases that hydrolyze UMP to uridine. Additional support for this interpretation is provided by the additive effects of the "$3" and NaF. Two other points require mention at this stage, regarding the identity of 5'-UMP as the first product of exonuclease action and the immediate precursor of UDP and UTP. The action of a polynucleotide phosphorylase, which has been detected in nuclei by Hilmoe and Heppel (20) and by Siebert et al. (21) and postulated by Harris (9), would liberate labeled UDP from the RNA. Under our conditions, UDP, if liberated, would have been readily phosphorylated to UTP (Table 3); this was not the pattern observed in Table 2. Furthermore, the action of polynucleotide phosphorylase should be facilitated by inorganic phosphate; we could detect no significant effect of 30 mM phosphate on rate of degradation of RNA or production of 5'-nucleotides (Table 1). One might also maintain with reason that 5'-UMP, UDP and UTP could be derived by rephosphorylation of uridine derived from dephos-

334

ROBERT B. HURLBERT e t

al.

phorylation of 2'(3')-UMP. We did not actually test for ability of these nuclei to phosphorylate uridine; uridine phosphokinase is active in rat liver, but is regarded as a cytoplasmic enzyme. We did, however, test whether a pool of uridine would diminish the recovery of RNA-uracil as UTP. In the experiment shown in Table 1, the apparent diminution probably was not significant, and in a separate experiment it increased the recovery of labeled UTP slightly, apparently by inhibiting the activity of the phosphatase attacking 5'-UMP.

Products of Degradation of Nuclear RNA Labeled with ~ldenine or a2p

Much of the above described work with uracil-labeled RNA was repeated by incubation of liver nuclei from rats labeled for ½ hr with all-adenine in vivo. The interpretations above were confirmed with the adenine label with regard to degradation of the rapidly labeled RNA and almost complete recovery of the adenine as 5'-nucleotides (ADP and ATP), yet a significantly different pattern of products suggested that ADP rather than AMP might be the primary product of the degradation. Table 4 shows the results obtained when nuclei were incubated in the standard medium, in the presence of "$3" fraction and in the presence of "$3" plus 0.04 M NaF. About 40 ~o of the labeled RNA was degraded in 15 min in the standard medium, about 32 ~o with "Sa" fraction added, and about 6 ~o in the presence of "Sa" and NaF. A rather striking difference in comparison with the uracil-labeled nuclei is observed: the zero time adenine-labeled nuclei contain about one-third of their total label as acid-soluble material, here shown by electrophoretic analysis to be 70 ~o ADP. Upon incubation, this initial ADP, and also the new products of degradation of the RNA, are rapidly phosphorylated to ATP. The conversion to ATP is somewhat more efficient when "$3" fraction is added, and is not diminished by NaF. There is very little indication that any 2'(3')-AMP is formed, even in the absence of "Sa" fraction, assuming that this product would be rapidly dephosphorylated to adenosine. Study of a2P-labeled nuclei. The relatively large amount of zero time nuclear ADP in these experiments was unexpected to us, and suggested some metabolic function of ADP peculiar to the adenosine nucleotides and the nucleus. This viewpoint was stimulated by the results of other experiments in which we attempted to account for all four nucleotide products by studying degradation of nuclear RNA previously labeled with 32p_phosphate. Rats were given injections of 3-4 mCi of a ZP-sodium phosphate, livers removed after 2 hr, and nuclei were prepared and incubated as usual. The acid-soluble fraction (including carrier nucleotide triphosphates) was

RNA

T U R N O V E R OF R A P I D L Y - L A B E L E D N U C L E A R

335

subjected to two-dimensional electrophoresis on 3 M M paper and D E A E paper, seeking primarily the nucleoside di- and triphosphates. The chromatographic results were not analytically precise enough to be reported in detail, although some features were clear. Much of the label in the acid-soluble fraction was phosphate, increasing from 20 ~o of the total at zero time to 40 ~ at 15 min. A m o n g the nucleotides at zero time, only spots in the A D P (about 45 ~ of the acid-soluble organic phosphates) and A T P (about 28 ~ ) regions were identifiable by autoradiography on X-ray paper. Following incubation of the nuclei for 15 min in the standard medium containing "$3" TABLE4.

PRODUCTS OF ADENINE-LABELED NUCLEAR

Incubation conditions and time (min)

Percentage of total 3H as cold PCA-soluble (%)

RNA

DEGRADATION

Percentage of cold PCA-soluble 3H as AR (%)

AMP (%)

ADP (%)

ATP (%)

Standard incubation 0 5 15

30 52 73

9 5 5

72 19 16

12 70 71

Plus "Sa" extract 0 5 15

29 45 61

9 3 2

71 16 10

12 77 80

Plus "S 3" + NaF 0 5 15

29 32 35

8 3 2

70 18 14

13 78 82

One rat received 750/tCi of all-adenine for ½hr. Nuclei were prepared, incubated under standard conditions, extracted and the cold PCA-soluble nucleotides were analyzed by electrophoresis as described in Methods. The content of 3H in the nuclear RNA at zero time was about 100,000 dpm per DNA. AR is adenosine; AMP is adenosine-5'-phosphate. fraction, the electropherograms could be seen to possess faint autoradiogram shadows corresponding to UTP, CTP and G T P (6-8 ~o of the total) with a large dominating A T P spot (46 ~ ) and a smaller A D P spot (18 ~o). It was not successfully established what percent of the label in the A D P and A T P was in the alpha phosphate position, leaving open the possibility that a considerable proportion was in the beta and g a m m a positions. While comparable levels of degradation of R N A uracil, cytosine and guanine moieties, with recovery as UTP, C T P and GTP, did appear to occur, it was clear that the metabolism of A D P and A T P by the nuclear preparations differed greatly from the metabolism of the other nucleotides.

336

ROBERT B. H U R L B E R T

et

al.

Further examination of the nuclear ADP pool In order to gain more perspective on the nature of the nuclear ADP, rats were labeled with 3Hadenine for ½ hr and liver nuclei were isolated, then extracted directly without delay or addition of reaction mixture. The cold PCA extracts of nuclei and of the cytoplasmic fraction were chromatographed on Dowex-1 (formate) columns (13), the amounts of AMP, A D P and ATP were determined, and the specific activities of the nuclear A D P and cytoplasmic ATP were measured. Table 5 presents the data: whereas 6 0 ~ of the cytoplasmic 3H-adenylates was in ATP, with 30 9/ooin ADP, the nuclear A D P represented about 75 ~o of TABLE 5. COMPARISON OF NUCLEAR AND CYTOPLASMIC ADENYLATES IN RAT LIVER LABELED WITH 3H=ADENINE FOR ½ HR

of cold PCA-soluble adenylates as AoMooP ADP Cytoplasm Nucleus

14

75

16V1

Specific activities of adenylates, dpm//2mole ADP

ATP

1.15 x 106 1.14 x 106

1.12 x 106

RNA-adenine

0.48 x 106

Two rats received 750 pc each of 3H-adenine, and ½hr later nuclei were prepared. The cytoplasmic supernatant was extracted with cold 0.4 N PCA. The nuclei in 0.05 M Tris (pH 7.6) were treated with phenol at 0°. The aqueous fraction was washed with ether and treated with cold 0.4 N PCA to obtain PCA-soluble nucleotides. The phenol phase was further extracted with 0.5 ~ sodium dodecyl sulfate in 0.1 M sodium acetate (pH 5.5) and 5 mMEDTA at 65° to obtain the rapidly labeled RNA fraction, designated "RNA-adenine" above, which was hydrolyzed with alkali and chromatographed to obtain 2'(3')adenylic acid. The nuclear cold PCA-soluble fraction and the rapidly labeled RNA fraction each contained about 1~ of the total cellular label. The cytoplasmic adenylates above were only about 50% of the total cytoplasmic label most of the rest of the label was in the column effluent and early peaks. Less than 8 % of the nuclear acid-soluble label was in these effluent and early peaks. the nuclear adenylates. The specific activities of the cytoplasmic ATP and nuclear A D P were the same. The amount of aH-adenylates in the nucleus was 1-2 ~o (in several experiments) of the total labeled adenylates in the cytoplasm. Although small, this percentage is several times the percent of cellular '4Clabeled uridine nucleotides found in the nucleus as uridine and uridine nucleotides (mainly UMP) in comparable experiments with orotic acid-labeled rats. In one experiment we extracted the rapidly labeled R N A specifically by a hot phenol procedure (5) following a cold phenol extraction at pH 7.6, and compared the specific activity of the R N A adenylic acid with the specific activity o f the nuclear ADP. After incorporation for ½ hr in vivo of

TURNOVEROF RAPIDLY-LABELEDNUCLEARRNA

337

3H-adenine into rat liver, the ratio of specific activities, RNA-adenylate/ADP, was 0.42. These data suggest the rapidly labeled RNA is in close equilibrium with the precursor pool of cytoplasmic ATP or nuclear ADP. Examination of Nuclear Nucleases

The foregoing data indicate that the rapidly labeled RNA is degraded by an endonuclease which is inhibited by rat liver cytoplasmic ribonuclease inhibitor and an exonuclease which is inhibited by NaF. This has been tested more directly by extraction, fractionation and chromatography of ribonuclease activities from these nuclei. The methods of Lazarus and Sporn (10, 19) were used, although some modification was required for our needs. The exonuclease appears to be the same as the one reported by these authors, namely, a 5'-nucleotide-forming enzyme which attacks at the T-OH terminal. The major endonuclease activity in our experiments is apparently the alkaline ribonuclease II of vertebrates (see classification by Barnard, 22) which attacks preferentially adjacent to pyrimidine nucleotides to form 3'-nucleotides. This is the nuclease controlled by the inhibitor in the "$3" fraction (18). We find another nuclear endonuclease, as reported by Lazarus and Sporn, to be a 5'-P former; this is very likely the nuclear alkaline endonuclease I described by Razzell (23) and Heppel (24). Thus far, we have not found polynucleotide phosphorylase activity in the nuclear extracts by these methods. DISCUSSION

In this paper we report in more quantitative terms a mechanism which may be surmised from the literature (14, 19, 25) and which has been previously reported, although considerably less quantitatively, for animal cells (9): that rapidly labeled nuclear RNA is primarily degraded to 5'-nucleotides which are readily rephosphorylated to triphosphates and reincorporated into new RNA. We have provided in isolated liver nuclei evidence that the rapidly labeled RNA is susceptible to both endonuclease and exonuclease activity. Because the 3'-P forming endonuclease activity is controlled in the isolated nuclei by cytoplasmic nuclease inhibitor (contained in the "$3" fraction), we presume that in vivo the same control occurs. The device which permitted the demonstration that nuclear RNA is "turned over" or cycled via the 5'-nucleotides was the use of added nucleotide kinase, also contained in the "$3" fraction, plus the use of methods which maximize RNA synthesis in isolated nuclei. The observations that RNA-uracil (8) and RNA-adenine are both in close equilibrium with their nucleoside triphosphate precursors (provided the rapidly labeled RNA is selectively extracted) provide further evidence of turnover or exchange between the pools of rapidly labeled RNA and the nucleoside-5'-phosphates. M

338

ROBERTB. HURLBERTet al.

More strictly speaking, our statements about mechanism of RNA degradation rest mainly on the rather complete evidence that 5'-UMP is the primary degradation product of RNA-uracil. We have shown that the mechanism of degradation of RNA adenine may be obscured by presence and metabolism of ADP in the nuclear preparations. At present, our evidence is inconclusive whether 5'-AMP or ADP is the primary degradation product of RNAadenine. In 1963 Harris (2, 9) presented data showing that ADP was a small, but significant, product of the degradation of HeLa cell RNA, and we have confirmed and extended this observation. On the basis of this and other evidence, he attributed the finding to a polynucleotide phosphorylase activity, and this would be the simplest interpretation of our data as well, although a highly active nuclear AMP kinase or contamination by mitochondrial activity might give the same result. It is quite possible that one-half or more of the nuclear ADP we and Harris observed is not derived from the rapidly labeled RNA, but is involved in some other independent metabolic function and for some reason is more effectively retained during isolation of nuclei than other nucleotides. Certainly the exonuclease of Lazarus and Sporn known to be present in the nuclei should liberate some 5'-AMP because this enzyme was originally tested and found most effective against poly A. Although our data do not support the thesis that polynucleotide phosphorylase is involved in release of RNA uridylate groups, the enzyme has previously been detected in nuclei (20, 21). It is conceivable that the polynucleotide phosphorylase acts primarily on the poly A sequences of the rapidly-labeled RNA. The purpose of the work described was to establish whether the synthesis and degradation of large amounts of unused RNA is efficient enough to be considered as part of a regulatory mechanism for selection of messenger RNA, and whether the known nucleases seem to be functioning as predicted under simulated in vivo conditions. Whether the nuclear nucleases thus far involved have regulatory properties which would be responsive to intracellular metabolic signals is not established yet. The alkaline endonuclease II with its cytoplasmic inhibitor might represent such a system, but is detrimental to the formation of 5'-nucleotides. The nuclear endonuclease I is compatible with the nuclear exonuclease I as far as the production of 5'-nucleotides is concerned, but thus far no specificities or inhibitors have come to light to suggest regulatory properties. In some analogy with other nucleases, the nuclear exonuclease might represent a regulatory system. Ribonuclease V, a 5'-nucleotide forming 5'-P-end-attacking exonuclease of E. coli, has been reported by Kuwano et al. (26) to degrade mRNA on the polysome; it is believed to be controlled in some way during initiation of translation by a number of ribosomes before beginning degradation of the messenger RNA by following the last ribosome. Kelley and Perry (27) have shown an exonuclease in mouse L-cell

TURNOVER OF RAPIDLY-LABELED NUCLEAR R N A

339

nucleoli which releases labeled 5'-UMP moieties from uridine-labeled nucleolar RNA. Presumably this nuclease is controlled at least in part by strategically located methyl groups on the ribose at the 45 S preribosomal RNA during the scissions which degrade the 45 S RNA to the 18 S and 28 S RNAs. The nuclear exonuclease I does not, however, appear to us to have great advantages for participation in a regulatory mechanism. For one thing, in attacking at the 3'-OH end, it can attack an RNA molecule only after it has been released from the DNA template or has been subjected to 5'-P-forming endonucleolytic scission. In isolated nuclei, the DNA-like RNA is degraded almost as fast as it is formed, suggesting an attack on the free 5'-P end. For another, this nuclease is presumably not dissuaded from attack by the poly A sequences believed to be present and retained on the 3'-OH informative end of rapidly labeled RNA (7). It would appear that knowledge of other nucleases or other factors is necessary to explain the controlled turnover of rapidly labeled nuclear RNA. SUMMARY

In order to study the mechanisms of degradation of "rapidly labeled DNA-like" or "heterogeneous nuclear" RNA, rat liver nuclei were labeled for ½ to 2 hr in vivo with [14C]-orotic acid, then incubated in unlabeled medium. About half of the labeled RNA was lost in 20 min, even under conditions which permitted new RNA synthesis. The products of degradation were mostly uridine, uridine 2'(3')-phosphate and unidentified fragments, unless an extract of rat liver, containing a 5'-nucleotide kinase and an inhibitor of alkaline ribonuclease II, was added. Under these latter conditions, up to 70 ~o of the rapidly labeled RNA uracil was recovered as UTP, and it was shown that the uracil was released as 5'-UMP units by an exonuclease previously described by Lazarus and Spurn. Nuclear RNA labeled with [3H]-adenine or [32p]-phosphate gave a different pattern of degradation; ADP was the primary product observed and was rapidly phosphorylated to ATP. Some of this ADP was associated with the nuclei as isolated and may represent some metabolic system other than RNA turnover. It also appeared possible, but was not confirmed, that polynucleotide phosphorylase was responsible for releasing 5'-AMP units as ADP from the rapidly labeled RNA. The results indicate that the rapidly labeled RNA is synthesized from and degraded to a pool of 5'-nucleotides in a cyclic process. It is postulated that the process is efficient enough to provide the basis for a post-transcriptional genetic regulatory system which selectively protects certain messenger RNA molecules among the "rapidly labeled DNA-like" population and allows the rest to be degraded.

340

ROBERT B. HURLBERT et al. ACKNOWLEDGEMENTS

This w o r k was initiated by institutional funds p r o v i d e d by the A m e r i c a n C a n c e r Society an d the J o h n Q. G a i n e s F o u n d a t i o n an d s u p p o r t e d directly by G r a n t N o . G-447 f r o m T h e R o b e r t A. W e l c h F o u n d a t i o n .

REFERENCES 1. J. E. DARNELL,JR., Ribonucleic acids from animal cells, BacterioL Rev. 32, 262-290 (1968). 2. H. HARRIS,H. W. Fisnr.~, A. RODOEP.S,T. SPENCERand J. W. WATTS,An examination of the ribonucleic acids in the HeLa cell with special reference to current theory about the transfer of information from nucleus to cytoplasm, Proc. Royal Soc. B 157, 177-198 (1963). 3. J. W. WATTS, Turnover of nucleic acids in a multiplying animal cell, Biochem. J. 93, 306-312 (1964). 4. R. W. SHEARERand B. J. McCARraV, Evidence for RNA molecules restricted to the cell nucleus, Biochemistry 6, 283-289 (1967). 5. R. SoEmo, M. H. VAUGHAN)J. R. WARNERand J. E. DARNELL)JR., The turnover of nuclear DNA-like RNA in HeLa cells, J. Cell BioL 39, 112-118 (1968). 6. R. SOEmOand J. E. DARNELL,A comparison between heterogeneous nuclear RNA and polysomal messenger RNA in HeLa cells by RNA-DNA hybridization, J. Cell. Biol. 44, 467-475 (1970). 7. G. P. GEOROmV, A. P. RYSKOV, C. COUTELLE, V. L. MANTIEVA and E. R. AVAKYAN, O n the structure of transcriptionalunit in mammalian cells,Biochim. Biophys. Acta 2.59, 259-283 (1972). 8. R. B. HURLBERT and V. R. POTTER, Nucleotide metabolism. I. The conversion of orotic acid-6-C ~* to uridine nuclcotidcs,J. Biol. Chem. 209, 1-21 (1954). 9. H. HARRIS, The breakdown of ribonucleic acid in the cell nucleus, Proc. Roy. Soc. B 158, 79-87 (1963). I0. H. M. LAZARUS and M - B. SPORN, Purification and properties of a nuclear exorbionuclease from Ehrlich ascites tumor cells,Proc. NatL Acad. Sei., U.S.A. 57, 13861393 (1967). 11. T. TAZ.AnASm,R. B. SWINTand R. B. HLrRLUERT,Synthesis of RNA in isolated nuclei of the Novikoff ascites tumor, ExptL Cell Res., SuppL 9, 330--344 (1963). 12. R. B. HORLU~Rr, E. G. MILLER and C. L. VAUGHAN,Control of RNA polymerase reactions in isolated nuclei and nucleoli, Advances in Enzyme Regulation 7, 219-233 (1969). 13. R. B. HURLBV.RT,Preparation of nucleoside diphosphates and triphosphates, Methods in Enzymology III, 785-805 (S. P. COLOWtCKand N. O. KAPLAN,eds.), Academic Press (1956). 14. J. W. WAaWS, The loss of rapidly labelled RNA from isolated HeLa cell nuclei, Biochem. J. 112, 71-79 (1969). 15. T. H. HAMILTON,Effects of sexual steroid hormones op genetic transcription and translation, pp. 56-92 in Basic Actions of Sex Steroids ', Target Organs, S. Karger, Basel (1971). 16. F.-L. Yu and P. F~ICELSON, Studies on the role of ammonium sulfate in nuclear transcription in vitro, Biochim. Biophys. Acta 272, 119-123 (1972). 17. E. HERBERTand V. R. POTTER, Nucleotide metabolism, VI. The phosphorylation of 5'-cytosine and guanine nucleotides by cell fractions from rat liver, J. BioL Chem. 222, 453-467 (1956). 18. J.S. ROa'H, Some observations on the assay and properties of ribonucleases in normal and tumor tissues, Methods in Cancer Research III, 154-237 (H. BuscH, ed.), Academic Press (1967).

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RNA

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19. M . B . SPURN, H. M. LAZARUS,J. M. SMITHand W. R. HENDERSON,Studies on nuclear exoribonucleases. III. Isolation and properties of the enzyme from normal and malignant tissues of the mouse, Biochemistry 8, 1698-1706 (1969). 20. R . J . HILMOE and L. A. HEPPEL, Polynucleotide phosphorylase in liver nuclei, J. Am. Chem. Sue. 79, 4810-4811 (1957). 21. G. SIEBERT,J. VILLAI.,OBOS,T. S. Ro, W. J. STEELE,G. LINDENMAYER,H. ADAMSand H. BuscH, Enzymatic studies on isolated nucleoli of rat liver, J. Biol. Chem. 241, 71-78 (1966). 22. E. A. BARNARD,Ribonucleases, Ann. Reo. Biochem. 38, 677-732 (1969). 23. W. E. RAZZELL, Phosphodiestcrases, Methods in Enzymology, VI, 249-252 (S. P. COLOWlCK and N. O. KAPLAN, ¢ds.), Academic Press (1963). 24. L. A. HEPPEL, Pig liver nuclei ribonuclease, pp. 31-36 in Procedures in Nucleic Acid Research (G. L. CANTONIand D. R. DAVIES,eds.), Harper & Row (1966). 25. M. F. SINGER and G. TOLBERT, Purification and properties of a potassium-activated phosphodiesterase (RNAse ID from E. coli, Biochemistry 4, 1319-1330 (1965). 26. M. KUWANO, D. SCHLESSINGERand D. APIRION, Ribonuclease V of E. coll. IV. Exonucleolytic cleavage in the 5' to 3' direction with production of 5'-nucleotide monophosphates, J. Molec. BioL 51, 75-82 (1970). 27. D. E. KELLEYand R. P. PERRY, The production of ribosomal R N A from high molecular weight precursors. II. Demonstration of an exonuclcase in isolated nucleoli, Biochim. Biophys. Acta 238, 357-362 (1971).

NOTE ADDED IN PROOF

Several types of experiments using nuclei prelabeled with 3H-adenine and prepared by the technique of G. Blobel and V. R. Potter (Science 154, 1662-1665, 1966) have shown conclusively that AMP rather than ADP is the major product released by degradation of the RNA. These nuclei contain much less zero-time ADP and less AMP kinase than the "isotonic" nuclei. We thus have evidence against the possibility that polynucleotide phosphorylase is quantitatively important in the intranuclear degradation of HnRNA (cf. also Y. P. See and P. S. Fitt, Biochem. J. 130, 355-362, 1972).