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) E )9 rI I .17. T 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.