Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 5009-5013, December 1974

Synthesis and Processing of Nuclear Precursor-Messenger RNA in Avian Erythroblasts and HeLa Cells (RNA turnover/regulation/complementary DNA/hemoglobin) GEORGES SPOHR, TEREZA IMAIZUMI*, AND KLAUS SCHERRER* Department of Molecular Biology, Swiss Institute for Experimental Cancer Research, CH-1011 Lausanne, Switzerland Communicated by Jacques Monod, September 23, 1974 ABSTRACT The kinetics of synthesis and turnover of (avoiding actinomycin D) are possible without impairing animal cell nuclear precursor-mRNA fractions all of RNA synthesis (3, 4). RNA was analyzed on exponential which, in the case of avian erythroblast RNA, are shown by gels, which allow the simultaneous analysis of specific complementary DNA hybridization to contain polyacrylamide globin mRNA sequences, were analyzed by exponential molecules over a MW range of 104 108 (11) as well as their polyacrylamide gel electrophoresis. Three metabolically sizing by comparison with internal MW standards. distinct size-fractions were characterized: (1) nascent precursor-mRNA (apparent molecular weight 5 to 20 X METHODS 106, approximate half-life 30 min), (2) intermediate-size and RNA was ex- precursor-mRNA (molecular weight 1 to 5 X 106, approxi- Immature duck erythrocytes were labeled mate half-life 3 hr), (3) small precursor-mRNA (molecular tracted by hot phenol (16) from purified nuclei as described weight 0.5 to 1.5 X 106, half-life more than 15 hr). Nascent previously (15). HeLa (human) cells (S strain) were grown in precursor-mRNA behaves kinetically as a precursor to the Jocklik modified Eagle's medium supplemented with 10% smaller precursor-mRNAs that accumulate in the nucleus, The were labeled in complete medium at a as well as to cytoplasmic mRNA; however, no stringent calf serum. cells proof can be given that the two smaller nuclear precursor- concentration of 20 to 30 X 10Q cells per ml (17). Sedimenta- mRNA fractions are direct physical precursors of func- tion analysis was performed in exponential sucrose gradients tional mRNA. In terms of total mass, more precursor- according to Noll (18). Electrophoresis on polyacrylamide mRNA accumulates in the nucleus than there is trans- gels was performed according to Mirault (11). Unless spe- lated mRNA in the cytoplasm. Globin mRNA of final size (9 S) does not accumulate in the nuclei of avian erythro- cified, the conditions of electrophoresis were: mixing volume, blasts. VI = 0.8 ml; gel diameter, d = 6 mm; length, 1 = 130 mm; 12 hr at 10 V/cm, 4°. Animal cells contain in their nuclei a class of nonribosomal For extraction of the RNA from the gels, 1.5-mm slices RNA having an apparent molecular weight (MW) in the were incubated with 300 ,Al buffer (0.01 M Tris*HCl, pH 7.4; range 1 to 30 X 106. This RNA was long suspected to be a 0.01 M NaCl; 0.1% sodium dodecyl sulfate) for 24 hr at 4°. precursor to mRNA (1-5 and reviews 6 and 7). Recently (8), The RNA was either precipitated with 2 volumes of ethanol, we have been able to provide conclusive evidence that sequences or lyophilized and then dissolved in a small volume. identical to globin mRNA can be found in the covalent pri- mary sequence of nuclear RNA of 106-107 MW from duck RESULTS erythroblasts. Hence, this RNA contains the coding informa- Estimation of pre-mRNA MW by gel electrophoresis may be tion for globin and represents a precursor to mRNA which we fallacious due to secondary structure. We determined on gels called pre-mRNA (8). The modalities of synthesis and pro- in aqueous medium an upper MW limit of 2 to 3 X 107 for cessing of this pre-mRNA are still poorly understood. If our nuclear RNA; in totally denaturing formamide gels no pre- evidence qualifies pre-mRNA as the informational precursor mRNA molecules with more than 1 X 107 MW could be to mRNA it still leaves open the question of whether or not it found (27). However, the aqueous medium allows a better represents the direct physical precursor. Thus, in order to separation of pre-mRNA fractions on gels; hence this tech- comprehend the mechanisms and the regulation of mRNA nique was adopted for the present analysis. synthesis, more knowledge concerning synthesis and turn- Demonstration of Specific Messenger Sequences in Giant pre- over of pre-mRNA is necessary. mRNA. To demonstrate that RNA molecules migrating in the Previous work concerned with the metabolism of pre- 106-107 MW range contain mRNA sequences and, hence, mRNA did not lead to unambiguous results due to various represent genuine pre-mRNA, total nuclear RNA from avian obstacles (3-5, 7, 9, 10). We report in the present publication erythroblasts was fractionated on gels. The RNA contained an investigation on synthesis and turnover of pre-mRNA in individual gel slices was eluted and hybridized with duck which attempts to circumvent some of these difficulties. In globin anti-messenger DNA (amDNA) as described pre- erythroblasts pulse-chase experiments by isotope dilution viously (8). Fig. 1 demonstrates that the globin mRNA se- Abbreviations: pre-mRNA, precursor to mRNA; amDNA, DNA quence is contained in 106-107MW RNA; smaller, processed complementary to messenger RNA (anti-messenger RNA); MW, molecules contain higher amounts of hybridizable RNA. molecular weight. This result was confirmed recently using polyacrylamide- * Present address: Institute de Biologie Moldculaire, Universite formamide gel electrophoresis carried out under conditions de Paris VII, Faculte de Paris VII, Faculte des Sciences (tour melting out the globin mRNA - amDNA hybrid (Imaizumi, 43), 2, place Jussieu, F-75 005 Paris, France. Spohr, and Scherrer, unpublished). Thus we conclude that the 5009 5010 Biochemistry: Spohr et al. Proc. Nat. Acad. Sci. USA 71 (1974)

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SUCE NUMBER FIG. 1. Globin mRNA sequences in erythroblast nuclear RNA fractionated by gel electrophoresis. Nuclear RNA was fraction- ated on 20.0-1.8% exponential gels according to Methods. The radioactivity of 25 Al of gel eluate was counted and 100 Ml were lyophilized, dissolved in 10 Al of bidistilled water, adjusted to contain in a final volume of 20 Ml 50 mM triethanolamine, 0.3 M NaCl, 0.5% sodium dodecyl sulfate, and 2500 cpm of globin amDNA, and hybridized at 650 for 42 hr. Digestion was at 450 0 20 40 60 0 20 40 60 for 40 min with S1 nuclease as described (8). 8H-labeled RNA was e SLICE NUMBER ® G SLICE NUMBER i totally eliminated by 0.3 N NaOH for 18 hr at 370 prior to tri- chloroacetic acid precipitation and measurement of hybridized FIG. 2. Comparison of nuclear pre-mRNA and polyribosomal amDNA(....). mRNA from HeLa cells by polyacrylamide gel electrophoresis. (A, A'), HeLa cells synchronized (to emphasize histone mRNA) by double thymidine block (26) were labeled after the second giant nuclear RNA of more than 106 MW contains specific thymidine block release for 3 hr with [;H]uridine (1 MCi/mi, 20-30 messenger sequences 4nd, hence, is pre-mRNA. Ci/mmol, 0.05 Mg/ml of actinomycin D) and the RNA was The Qualitative and Quantitative Size-Spectrum of Nuclear phenol-extracted as described previously (15). The same amounts pre-mRNA and Its Comparison to Cytoplasmic mRNA. In were analyzed on two different gel concentrations. (B), Unsyn- HeLa cells incubated with low doses of actinomycin D, which chronized HeLa cells were labeled for 6 hr with [3H]uridine (0.66 selectively inhibits ribosoinal RNA synthesis, a quantitative ACi/mi, 0.5 AM uridine, 0.05 Mg/ml of actinomycin D), poly- determination of the steady-state population of pre-mRNA is ribosomes were prepared by sedimentation on sucrose gradients, possible. Fig. 2A and A' show the size distribution of pre- and the RNA was phenol-extracted (15). (A), total cell RNA: 6.0- mRNA and mRNA in HeLa cells labeled to nu- 1.8% gel, 6 hr at 120 V (A'); total cell RNA: 12.5-2.5% gel, 4.5 synchronized hr at 120V; (B), polyribosomal mRNA: 15-2% gel, 9 hr at 120 V. clear steady-state in S phase. For comparison, Fig. 2B shows Solid line, broken line, [3H]uridine incorporation. the steady-state mRNA population isolated from purified poly- A2M; ribosomes of an unsynchronized culture. This mRNA pop- ulation shows the expected size-spectrum in the 105-106 MW nuclei (Figs. 3, 4, and 6). The question must be raised if any range. It is evident that among the total cell RNA the amount mRNA in its final size is present in nuclei. of radioactivity in the size-range characteristic of mRNA is On the basis of these experiments we conclude that in very small compared to that of pre-mRNA. In the cyto- highly differentiated as well as in undifferentiated animal plasm, on the contrary, the contribution of molecules with cells the bulk of RNA involved in mRNA synthesis (pre- MWs of more than 2 X 106 is insignificant (compare Fig. 213 mRNA) is constituted of molecules with molecular weights in and 3B). Thus, less than 15% of the total cellular pre-mRNA excess of 106 MW. Since these molecules are confined to the and mRNA migrate with the characteristics of messenger- nucleus we are led to the rather surprising finding that the size molecules and could possibly represent functional mRNA. bulk of messenger-related RNA in animal cells is localized in Fig. 3 relates the result' of a similar analysis carried out in the nucleus. erythroblasts, which devote 80-90% of their protein syn- Rate of Synthesis and Decay of pre-mRNA. We reported thesis to hemoglobin (3, 4). In these nondividing cells rRNA previously an analysis of pre-mRNA turnover in HeLa cells synthesis cannot be inhibited selectively but it is very low. carried out by actinomycin D chase (5). Since in HeLa cells Although in these cells 9S globin mRNA amounts for about pulse-chase experiments by isotope dilution are impossible, 30% of the polyribosomal mRNA (compare Fig. 3B, and ref. we confined our present analysis to duck erythroblasts. Such 15), the nuclear pre-mRNA shows essentially the same size- experiments are valid, since double labeling experiments re- spectrum as that observed in HeLa cells. The heterogeneity of veal that [3H]adenosine incorporation and, hence, RNA syn- the size-spectrum of pre-mRNA is not surprising, since about thesis continues unimpaired after dilution of [l4C]uridine 10% '(representing more than 103 average-size transcrip- with the unlabeled ribonucleoside (Spohr, unpublished). tional units) of the erythroblast genome is transcribed in this Duck erythroblasts were labeled continuously for various cell population in which all maturation forms of erythrocytes time periods ranging from 20 to 220 min. Nuclei were purified are represented (2). and their RNA was extracted by hot phenol and analyzed on It is interesting to note that no 9S peak can be observed exponential gels. The electrophoretic pattern (Fig. 4) shows among the RNA molecules extracted from highly purified that throughout the entire incubation period the largest Proc. Nat. Acad. Sci. USA 71 (1974) Synthesis and Turnover of Precursor-mRNA 5011

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FIG. 3. Comparison of nuclear pre-mRNA and polyribosomal I mRNA from duck erythroblasts by polyacrylamide gel electro- phoresis. Immature duck erythrocytes (1 ml of cells in 3 ml of anemic duck plasma) were labeled for 80 min with [3H]uridine (100 uCi, 20-30 Ci/mmol), nuclei were purified, and polyribo- somes were prepared by sedimentation on sucrose gradients and their RNA was phenol-extracted as described previously (15). Electrophoresis was according to Methods. (A), Nuclear RNA (20-1.8% gel); (B), polyribosomal RNA (20-2.2% gel). ( ), A260; (0 0), [3H]uridine incorporation; (0- - -0), 18S [14C]- 2040 60 80 20 40 60 80 rRNA marker. SUCE NUMBER SLICE NUMBER FIG. 4. Synthesis of nuclear pre-mRNA in duck erythroblasts. fraction of the labeled pre-mRNA migrates in the zone cor- Immature duck erythrocytes (1 ml of cells in 6 ml of anemic duck responding to molecules of more than 5 X 100 MW (45 S). plasma) were labeled with [3H]uridine (125 ,uCi, 27 Ci/mmol) for After 220 min the fraction of labeled molecules with molecular 20 min, (A); 40 min, (B); 80 min, (C); and 220 min, (D). The weight less than 5 X 106 MW is higher than observed after 40 nuclear RNA was prepared and analyzed by gel electrophoresis as and 80 min. described in Methods; 20-1.8% gels; ( ), A2M; (@-), [3H]- On the basis of qualitative observations three fractions uridine incorporation. were defined to evaluate quantitatively the radioactivity in the nuclear pre-mRNA: (1) molecules with apparent mo- D or by a 103-fold dilution of the [3HJuridine with the un- lecular weight greater than 5 X 106 (45 S) corresponding to the labeled ribonucleoside. Fig. 6 relates the qualitative pattern nascent RNA [as we defined earlier, (2-4) "nascent RNA" re- of such pulse-chase experiments. It is evident that the mole- lates to completed molecules immediately after release from cules of highest molecular weight, the nascent pre-mRNA, the chromatin and not to growing molecules-upon birth the decay at the fastest rate, whereas, the intermediate-size and baby is completed!], (2) an intermediate-size RNA fraction small molecules are relatively more stable. This observation containing molecules with about 1 to 5 X 106 MW (26 S-45 S), is confirmed by the quantitative evaluation given in Fig. 7. and (3) the small MW fraction containing molecules with less .69- than 106 MW (less than 26 S). '0 The relative amounts of label in these fractions were cal- A TOTAL x B NUCLEAR RNA C INDIVIDUAL culated and normalized. Fig. 5 relates the kinetics of uridine CELL LYSATE 6- FRACTIONS incorporation into whole cell (Fig. 5A) and nuclear RNA 0 OF pre-mRNA x5 z 5- oF= (Fig. 5B) and the relative amount of radioactivity in the 0 dF E 4- three size classes defined above (Fig. 5C). It can be observed 4 c 15C * >45S RNA pool is labeled into steady-state after 3- UNLABELED that the nascent URIDINE LU 80 min; the intermediate-size and small RNA 10Co approximately 2- F 2- F ---<45S classes do not reach steady-state at that time. However, w5s >26S Q 1- o 1- CHSE their labeling kinetics are bi-modal with a fast initial and a r AcNOMYCIN <26S slow subsequent rate. These kinetics are consistent with the 1.2 3 4 56 7_8 E 1 5 6 40_120.200 280 presence in this size class of rapidly labeled growing chains HOURS Co HOURS MIN and/ of molecules resulting from processing of nascent pre- FIG. 5. Kinetics of [3Hluridine incorporation into whole duck mRNA. This interpretation is corroborated by the qualitative erythroblasts, nuclei, and purified nuclear RNA of different size observation (Fig. 4) that the RNA molecules of the heaviest classes. Cells were labeled as described in Fig. 4. (A) Whole cell: size class are not prevalent after 20 min of labeling but only trichloroacetic-acid-precipitable radioactivity was determined in aliquots of cell lysates and normalized relative to the absorbance after 40 or 80 min. This is not surprising if one recalls that the sulfate of 5 X 106 MW lasts at 260 nm. (B) Purified nuclei: after sodium dodecyl lysis, synthesis of a pre-rRNA molecule the acid-precipitable radioactivity was determined in aliquots and about 2.3 min (20), and pre-mRNA is synthesized at a rate of normalized relative to the absorbance at 260 nm. (C) Nuclear about 25 nucleotides per sec (30). Hence, molecules of the size RNA: purified nuclei were phenol extracted, the RNA was of 2 X 107 MW would need up to 10 min to be completed. analyzed on gels as shown in Fig. 4, and the radioactivity in three In consequence duck erythroblasts were labeled for up to migration zones corresponding to >45 S, from 45 S to 26 S, and 80 min prior to the induction of a chase by either actinomycin <26 S was computed and normalized. 5012 Biochemistry: Spohr et al. Proc. Nat. Acad. Sci. USA 71 (1974)

45S "8 140 A, B, i URIDINE CHASE 6000 -120 -100 A ------26S 4000 80 60 100o 2000 I7 40 IL -20 Q 80- . <45S 0- I N-26S E E jTOTAL C) w L 40- z z 0 - aX z-,45S IC- *20- Ip 0 80 180. I 240 MIN CHASE 1400 A3 1200 FIG. 7. Kinetics of decay of nuclear pre-mRNA in different 1000 size classes. Immature erythrocytes were labeled with ['H]uridine 800 I'I as described in Fig. 6, and a chase was induced after 80 min of 600 (A) unlabeled uridine, or (B) actinomycin D 400 labeling with either "-i4XI 200 (100 pg/ml). Nuclear RNA was prepared and analyzed by gel electrophoresis (20-1.8% gels). The radioactivity incorporated 20 40 60 80 20 40 60 80 in the three classes of RNA (>45 S, 45 S-26 S, <26 S) was com- SLICE NUMBER SUCE NUMBER puted, normalized on a cell basis, and expressed as percentage of FIG. 6. Decay of nuclear RNA in duck erythroblasts during the radioactivity present in each class at the beginning of the a chase by unlabeled uridine. Immature duck erythrocytes chase. (A), chase with unlabeled uridine; (B), chase with actino- (1 ml of cells in 5 ml of anemic duck plasma) were labeled for 40 mycin D. min (Al) or 80 min (B,) with ['H]uridine (120 pCi, 27 Ci/mmol). Unlabeled uridine (5 mM final concentration) was added to As shown previously (8), and pointed out recently by Bish- separate samples after 40 min (A, and As) or 80 min (B2 and B,) of op's group (29), most of the nuclear globin pre-mRNA is labeling and the cells were incubated for a further 40 (A2, B2) or found in 4 to 6 X 105 MW RNA, smaller than the nascent 220 (A,, B,) min. Nuclear RNA was extracted and analyzed pre-mRNA which represents the primary transcription prod- electrophoretically according to Methods (20-1.8% gels). uct. In fact, we demonstrate the existence of three classes of (@-@), [3H]uridine incorporation; (....), 18S ['4C]rRNA marker. nuclear pre-mRNA. Nascent pre-mRNA (5 to 20 X 106 MW) is synthesized and decays most rapidly (half-life 20-30 min); The fastest decaying nascent pre-mRNA molecules decay intermediate size pre-mRNA (1 to 5 X 106 MW) is consid- small with a half-life of 20-30 min, the intermediate-size class (1 to erably more stable (half-life approximately 3 hr) and to 15 X 105 MW) is quite stable (half-life of 5 X 10 MW) decay with a half-life of the order of 3 hr, pre-mRNA (1 whereas the small size-class is fairly stable, with a half-decay more than 15 hr). Computations show that these fractions the total time of 15 hr or more. Visibly, actinomycin D leads to an correspond to respectively 55%, 30%, and 15% of under-estimation of the half-lives of the smaller pre-mRNA. nuclear pre-mRNA. All contain globin-specific sequences, We conclude that in avian erythroblasts three distinct which distribute in a relation of 12% to 25% to 63%, re- relative to classes of pre-mRNA exist, which are characterized by their spectively. This inverted distribution of overall molecular weight and structure as well as their intrinsic sta- specific pre-mRNA reflects not only the length of pre-mRNA of bility. Although there is some cross-contamination of the relative to mRNA, but also the qualitative pleiotropy pre- RNA molecules of different stability in the different size mRNA synthesis, as well as the more rapid processing and classes, the three characteristic decay rates speak strongly in export of expressed versus nonexpressed transcription prod- mRNA is ob- favor of the reality of these classes as forms of pre-mRNA ucts. No accumulation in the nucleus of 9S metabolism. The analysis of HeLa cell pre-mRNA by actino- served, although in erythroblasts 30% of polyribosomal mycin D chase led to similar observations (5). mRNA is globin mRNA; however, large amounts of relatively stable nonglobin pre-mRNA do accumulate in the nucleus. DISCUSSION The mobility of RNA in gels is dependent on structure as be at- These results give additional evidence for the existence of well as on size; hence, no absolute MW values can nuclear precursors to globin mRNA of MW up to 5 X 106 in tributed to the pre-mRNA classes. The nascent pre-mRNA erythroblasts. Elsewhere we will show (Imaizumi, Spohr, class, however, behaves during electrophoresis as the largest and Scherrer, unpublished) that in totally denaturing me- molecules and hence could represent the physical precursor of dium (27) the qualitative electrophoretic pattern of globin the smaller molecules. The same conclusion can be drawn known pre-mRNA is the same as that presented here (Fig. 1). This from kinetic observations. In fact, one of the first fully confirms our earlier work with dimethyl sulfoxide gra- properties of pre-mRNA concerned its instability (3, 4, 12, 22). dients (8), results which were questioned in a recent review In respect to an old controversy relating to a possible (28) by (unauthorized) erroneous reference to unpublished size shift during the initial decay of pre-mRNA (compare refs. work done in association with our laboratory. However, the 2, 3, 4, and 5 versus 9), the present data give clear evidence emphasis of the work presented here concerns rather prop- that the largest nascent RNA class disappears rapidly upon erties of overall pre-mRNA, which represents, since about a chase. However, since nascent and intermediate size pre- no ab- 10% of the duck genome is transcribed (2), an estimated mRNA largely overlap on gels, only a relative and 10'-104 transcription units of the globin pre-mRNA size. solute shift of label from the first to the second class can be Proc. Nat. Acad. Sci. USA 71 (1974) Synthesis and Turnover of Precursor-mRNA 5013

observed. Nevertheless, since the nascent .pre-mRNA pool Although this model is plausible, the present data do not reaches steady-state rapidly when the intermediate size class allow us to definitively exclude that unknown information continues to be labeled linearly (after a short exponential processing steps occur between pre-mRNA and mRNA and phase probably due to overlapping growing and nascent that, hence, pre-mRNA is not a direct physical precursor. molecules) we conclude that the former pool must feed the The observations concerning synthesis and processing of the latter. adenylylated fraction of pre-mRNA (7) are essentially con- The question may be asked whether labeling kinetics of sistent with the results reported here, and are subject to nascent pre-mRNA are consistent with its role as precursor to similar limitations (compare discussion in refs. 24, 25). cytoplasmic mRNA. The specific activity of a pre-mRNA population synthesized at a constant rate and decaying ac- We thank Heidi Diggelmann for preparing amDNA, Anne- cording to a first-order reaction with a half-life of ti1, follows a Cecile Bussard and Sylvia Schmidlin for their excellent technical assistance, and Pierre Dubied and Jacqueline Villa for help during curve of the type: preparation of the manuscript. We thank also Drs. N. Acheson and R. Eisenmann for critical reading of this manuscript. This N =No [1-exp (_In 2 * t),[] investigation was supported by the Swiss National Foundation (Grants no. 3.613.71 and 3.829.72) where is the specific activity at steady-state (time t = oo). No 1. Scherrer, K. & Marcaud, L. (1965) Bull. Soc. Chim. Biol. 47, When this pre-mRNA is processed into mRNA according to a 1697-1713. first-order reaction the specific activity S of the mRNA will 2. Scherrer, K. & Marcaud, L. (1968) J. Cell. Physiol. 72, follow the equation: 181-212. 3. Scherrer, K., Marcaud, L., Zajdela, F., London, I. M. & Gros, F. (1966) Proc. Nat. Acad. Sci. USA 56, 1571-1578. = dto2esNo 1 -exp (- t-*t] [2] 4. Scherrer, K., Marchaud, L., Zajdela, F., Breckenridge, B. & Gros, F. (1966) Bull. Soc. Chim. Biol. 48, 1037-1075. Integration of 2 leads to 5. Scherrer, K., Spohr, G., Granboulan, N., Morel, C., Gros- claude, J. & Chezzi, C. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 539-554. S = KNot-KNo ln2 1-exp (- . t)] [3] 6. Darnell, J. E. (1962) Bacteriol. Rev. 32, 262-290. 7. Weinberg, R. A. (1973) Annu. Rev. Biochem. 42, 329. H. & K. Proc. can as the result of 8. Imaizumi, T., Diggelmann, Scherrer, (1973) The labeling of the mRNA be considered Nat. Acad. Sci. USA 70, 1122-1126. a linear and an exponential term. For t > 2t,/, the exponential 9. Penman, S., Vesco, C. & Penman, M. (1968) J. Mol. Biol. term varies barely and S follows essentially linear labeling 34, 49-60. kinetics. The graphical extrapolation of the linear part of S 10. Soeiro, R. & Darnell, J. E. (1967) J. Cell Biol. 39, 112. (t) gives-by first approximation-an intercept with t close to 11. Mirault, M.-E. & Scherrer, K. (1971) Eur. J. Biochem. 23, 372-386. tl/,. In other words, the assumption that the nuclear pre- 12. Scherrer, K., Latham, H. & Darnell, J. E. (1963) Proc. Nat. mRNA with a half-life of 20 min is a precursor to a cyto- Acad. Sci. USA 49, 240-248. plasmic mRNA implies that the mRNA appears with a lag of 13. Weinberg, R. & Penman, S. (1969) Biochim. Biophys. Acta 20 min approximately, when measured for t > 2tl/,; this is in 190, 10-29. with results 14. Rein, A. & Penman, S. (1969) Biochim. Biophys. Acta 190, accordance experimental (3, 4, 9, 15). 1-9. The interpretation of the intermediate size pre-mRNA is 15. Spohr, G., Kayibanda, B. & Scherrer, K. (1973) Eur. J. even more complex. Since in erythroblasts it includes the Biochem. 31, 194-208. mRNA sequences (8) and, in HeLa cells, contains qualita- 16. Scherrer, K. (1969) in Fundamental Techniques in Virology, New tively the same RNA classes as the nascent pre-mRNA (5), eds. Habel, K. & Salzmann, N. P. (Academic Press, York), pp. 413-432. we must conclude that it contains two metabolically distinct 17. Spohr, G., Granboulan, N., Morel, C. & Scherrer, K. (1970) fractions: (1) the cleavage products of nascent precursors to Eur. J. Biochem. 17, 296-318. mRNA types which are almost instantaneously further pro- 18. Noll, H. (1967) Nature 215, 360-363. cessed and exported to the cytoplasm-allowing an apparent 19. Jelinek, W. & Darnell, J. E. (1972) Proc. Nat. Acad. Sci. and cyto- USA 69, 2537-2541. first-order kinetic relation of nascent pre-mRNA 20. Greenberg, H. & Penman, S. (1966) J. Mol. Biol. 21, 527- plasmic mRNA-and (2) other pre-mRNA types which are 535. stored in the nucleus where they decay slowly with a half-life 21. Perry, R. P. (1962) Proc. Nat. Acad. Sci. USA 48, 2179- of 3 hr. This view is consistent with the observation that, in 2186. HeLa cells, the intermediate-size pre-mRNA (as well as the 22. Harris, H. (1963) Progr. Nucl. Acid. Res. Mol. Biol. 2, 19. 23. Attardi, G., Parnas, H., Hwang, M. & Attardi, B. (1966) J. nascent class) represents a qualitative spectrum of RNA se- Mol. Biol. 20, 145-182. quences 10 times as large as that of cytoplasmic mRNA (ref. 24. Scherrer, K. (1973) Karolinska Symp, 6, 95-129. (Acta 5, Table 1). Endocrin. (Kbh) suppl. 180) Thus, corroborating earlier propositions (5), we can derive 25. Scherrer, K. (1973) Oholo Symp. 1973; Advan. Exp. Med. a model of processing where the nascent pre- Biol. 44, 169-219. pre-mRNA 26. Fan, H. & Penman, S. (1970) J. Mol. Biol. 50, 655-670. mRNA gives rise to intermediate-size molecules which are 27. Spohr, G., Mirault, M.-E., Imaizumi, T. & Scherrer, K. either rapidly processed and exported to the cytoplasm, or (1974) Eur. J. Biochem., in press. stored in the nucleus for prolonged times in the form of inter- 28. Davidson, E. & Britten, R. J. (1973) Quart. Rev. Biol. 48, mediate-size or small pre-mRNA. They may represent inter- 565-613. 29. MacNaughton, M., Freeman, K. B. & Bishop, J. 0. (1974) mediate steps within the scheme of cellular information Cell 1, 117-125. transfer from gene to phenotypic expression ("cascade regu- 30. Egyhazi, E. (1974) Symp. Biochem. Cell Nucleus, Proc. 9th lation") discussed elsewhere (2, 5, 24, 25). FEBS Meeting, Budapest.