Dec 22, 1983 - '15S' (Spohr et al., 1974; Ross and Knecht, 1978; Curtis et al., 1977) molecules, and by .... blots were essentially as described (Thomas, 1980).
The EMBO Journal vol.3 no.3 pp.491-495, 1984
Altered globin gene transcription pattern and the presence of a 7 -8 kb otA globin gene transcript in avian erythroblastosis virus-transformed cells
A.Therwath, G.Mengod1 and K.Scherrer* Institut Jacques Monod, Universite Paris VII, Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France 'Present address: Biozentrum der Universitat Basel, CH-4056 Basel, Switzerland *To whom reprint requests should be sent Communicated by K.Scherrer
Immature chick erythroid cells transformed by avian erythroblastosis virus (AEV) display an altered pattern of globin gene transcription leading to the abortive phenotypic expression of such transcripts. Detectable adult globin gene-specific RNA sequences, confined exclusively to the nucleus, are uniquely of the aA type. The aA globin-specific sequences occur in transcripts 7-8 kb long from which the 5' contiguous aD gene product is absent, and also in fragments smaller than 9S globin mRNA. The implication of this observation for schemes of post-transcriptional regulation of gene expression and viral transfonnation are discussed. Key words: avian erythroblastosis virus/globin genes/premRNA/abortive processing
which is fully degraded in the nucleus of AEV-transformed cells, (ii) the change in transcription pattern with respect to the type of the globin gene transcribed in AEV-transformed versus normal cells and (iii) identification and characterization of the transcription products; this latter question was approached by two independent methods, which gave essentially the same result. Our results indicate that, in chick erythroblasts transformed in vivo by AEV, aD and (3 globin gene products are below the level of detection among the nuclear RNAs; in the case of the aA gene, processing is perturbed leading to the accumulation of a globin mRNA-containing transcript of 7.3 kb which is abortively metabolized. Results Abortive transcription of the globin genes and titration of the amount of globin pre-mRNA Several independent experiments (not shown) involving hybridization of globin cDNA to AEV-transformed cell RNA confirmed our previous results (Therwath and Scherrer, 1978). We find, on the average, 100 molecules per transformed cell containing globin mRNA sequences. These molecules are exclusively in the cell nucleus with no detectable trace in the cytoplasm. The RNA molecules containing globin specific sequences are degraded within the nucleus: they are eliminated probably as a result of an aberrant or abortive processing event. This general conclusion is valid for cells isolated from infected birds: different results are obtained with bone marrow cells transformed in vitro (Therwath and Scherrer, 1982). Determination of the type of the globin genes transcribed by dot-blot hybridization We reported previously that when a mixture of a and A globin cDNA is reacted with nuclear RNA of AEV-transformed erythroblasts, only about half of this cDNA was annealed. This observation led us to analyse the RNA transcripts in these cells with cloned cDNA probes, using the dot-blot hybridization technique, which allows rapid determination of the relative concentration of nucleic acids in a mixture, as well as the extent of sequence homology between related RNAs and/or DNAs (Kafatos et al., 1979). Figure 1 shows that clone purified globin cDNAs detected their RNA homologues on the filters and failed to hybridize to control filters where yeast tRNA was spotted. Thus, each of the specific aA, aD or (3 probes detected its complementary sequence in the unfractionated globin 9S mRNA as well as in the nuclear RNA of normal erythroblasts. However, when nuclear RNA of the AEV-transformed cells (AEV cells) was analysed, the only positive signal obtained was with the aA cDNA probe. Thus, in the AEV-transformed chick erythroblast, transcripts of the oaA gene are detectable, whereas RNA corresponding to the aD and ,B globin genes is not present in measurable amounts. -
Introduction Transformation of vertebrate cells by RNA tumor viruses leads to a large number of phenotypic alterations including changes in the synthesis of discrete groups of proteins. We have previously reported the absence of any detectable hemoglobin synthesis in the avian erythroblastosis (AEV-Paris) transformed chick erythroblasts cultured in vitro for several months. However, globin gene-specific RNA sequences are found, but they are confined exclusively to the nuclear compartment. The failure of the phenotypic expression of the globin genes may result from aberrations at the processing level of globin pre-mRNA (Therwath and Scherrer, 1978), which we define as any specific gene transcript containing mRNA sequences, irrespective of whether or not such molecules are destined to be processed into functional mRNA (Scherrer et al., 1979; Scherrer, 1980). The number of globin mRNA-containing transcripts per transformed cell was calculated to be 100. This number is at least 20-fold less than that estimated for normal erythroblast nuclear RNA (Imaizumi et al., 1973). Furthermore, the RNA:globin cDNA hybridization data obtained previously indicate that apparently not all of the three adult globin gene transcripts are detectable in the nuclear RNA of AEVtransformed cells (Therwath and Scherrer, 1978). Analysis of the globin genes at the DNA level revealed an unaltered general organization but also the presence of additional methylation sites in and around the aD and : globin genes in the AEV-transformed cell as compared with normal cell DNA (Marcaud et al., 1981). The results presented here deal with three issues: (i) the abortive transcription of the globin genes into pre-mRNA, -
© IRL Press Limited, Oxford, England.
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Fig. 2. Optical density and radioactivity profile of AEV cell nuclear RNA. [3H]Uridine-labelled (45 min) total nuclear RNA was fractionated by exponential gradient polyacrylamide gel electrophoresis in pure formamide at 45°C. (------) shows the A280 nm scan of the electrophorogram and ( shows the profile of the radioactivity incorporated in the RNA. rRNAs electrophoresed in parallel gels served as mol. wt. markers.
Determination of globin pre-mRNA size by high temperature gel electrophoresis To characterize the abortively processed globin pre-mRNA by mol. wt., total nuclear RNA from AEV cells labelled with tritiated uridine for 45 min was fractionated in conditions allowing complete denaturation of all RNA secondary struc492
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RNA recovered from each of the fractions/zones was reacted with 4000 c.p.m. of [3H]dCTP-labelled globin cDNA in a hybridization reaction. The annealing reaction was carried out to a Crot value of 100 and contained, on average, a 10-fold excess of globin cDNA compared with the globin pre-mRNA sequence. The amount of the radioactivity recovered in the RNA-cDNA hybrid after SI treatment, for each of the RNA fractions analysed, is represented by the histogram.
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Fig. 3. Histogram showing the titration of globin pre-mRNA in AEVtransformed chick erythroblasts. Labelled nuclear RNA from AEVtransformed chick erythroblasts was fractionated on polyacrylamideformamide gels as described in Figure 2. The gel was cut into 10 equal slices and the RNA eluted from each slice. Equal amounts (0.15 Itg) of the
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ture (Spohr et al., 1976). Figure 2 shows the A280 nm scan of a gel electrophorogram, as well as the radioactivity profile determined in individual size fractions of nuclear RNA. Some of the nuclear RNA seems to be 28S and 18S rRNA (markers in parallel gel), but > 700/o of the radioactivity is present in the zone of rapidly turning over and newly synthesized molecules (cf., review by Scherrer et al., 1979) larger than 28S rRNA. In view of previous studies, these profiles indicate that the high mol. wt. RNA was largely intact having survived the extensive purification procedures. The distribution of globin mRNA sequences in the fractionated nuclear RNA of AEV cells was determined. The technique for RNA extraction from the gel and elimination of acrylamide proved to be efficient and independent of mol. wt. (Reynaud et al., 1980). Judged from absorbance and radioactivity measurements of the eluate, up to 75% of the radioactivity was recovered. In all, -350 /zg of RNA was fractionated on the gels, which were scanned at A280 and sectioned into 10 equal slices. Equal amounts of RNA recovered from pools of each slice fraction were annealed to labelled globin cDNA to reach a Crot value of 100, in at least 10-fold cDNA excess relative to globin mRNA. The histogram in Figure 3 gives the results of these titration experiments: globin mRNA sequences are contained in two distinct populations of nuclear RNA. A 7-12 kb pre-mRNA (3-4 x 106 mol. wt.) is separated from the low mol. wt. fraction, which culminates in molecules smaller than 0.6 kb globin mRNA. Globin mRNA sequences were not detectable as peaks in the region of the usual 1.5-kb and 3.8-kb precur-
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Fig. 4. Determination of mol. wts. of globin pre-mRNA in normal and AEV-transformed chick erythroblasts by RNA-blot hybridization. Chick reticulocyte nuclear RNA recovered from the exclusion volume of Biogel A 50 m (lanes 1 and 3, 30 Ag/slot), total nuclear RNA from AEVtransformed chick erythroblasts (lanes 2 and 4, 30 jg/slot), globin 9S mRNA (1.5 pg/slot), chick ribosomal 18 and 28S RNA (2 rg/slot) and avian sarcoma virus (ASV) 35S RNA (2 ytg/slot) was glyoxalated and fractionated by electrophoresis on a 10Go agarose gel and transferred to nitrocellulose sheets as described in Materials and methods. The bound RNA was reacted with 5 x 106 c.p.m./cm2 of 32P-labelled globin chainspecific probes (sp. act. 3-5 x 109 c.p.m.) for 36 h and the hybrids visualized after autoradiography. 9S globin mRNA, ribosomal 18 and 28S RNA and the ASV 35S RNA served as mol. wt. markers and was visualized after ethidium-bromide intercalation followed by u.v. illumination (marker lanes not shown). Lanes I and 2 were exposed to the aA globin gene-specific probe, and lanes 3 and 4 to the caD genespecific one.
sors described by Spohr et al. (1974) and by Bastos and Aviv (1977). Even when a further 10-fold excess of RNA in the 4-7 kb range was annealed to globin cDNA, we failed to detect any hybridization (data not shown). Since the AEV cell nuclear RNA contains 100 globin pre-mRNA molecules (Therwath and Scherrer, 1978), we can estimate from the gels that there are 20 globin pre-mRNA molecules in the 7-12 kb range; this is comparable with the amount of premRNA with this mol. wt. in normal erythroblasts (Reynaud et al., 1980). The remaining 80 molecules are heterogeneous, smaller than marker 18S rRNA and culminate in a peak smaller than 9S globin mRNA. By contrast, in the normal cell there are 1000 ' 15S' globin pre-mRNA molecules (Reynaud et al., 1980; Spohr et al., 1974). Taken together, these results indicate the existence in the AEV cell of a larger than 7-kb globin gene transcript in amounts almost comparable with those in the normal cell, -
and the absence of the discernible, specific '15S' globin premRNA widely described in the literature (loc. cit.). Determination of mol. wts. of globin pre-mRNA in normal and AEV-transformed chick erythroblasts by RNA-blot
hybridization We sought to confirm these titration-hybridization results by Northern blotting. The extremely low amount of globin premRNA in total nuclear RNA (0.003%), the large size expected for the globin pre-mRNA ( - 10 kb) and the limits of the specific activity of the cDNA probe, required that transfer of RNA from the gel to nitrocellulose sheets be complete. This was achieved by controlled nuclease digestion of the high mol. wt. molecules in situ prior to transfer (Therwath and Strauss, 1981). To optimize the specific activity of the probe, globin mRNA (previously purified by hybridization to cloned globin cDNA) was reverse transcribed to yield cDNAs with specific activity of 3-5 x 109 c.p.m. (nick-translation of the cloned DNA gave an 8 times lower specific activity). Figure 4 shows the autoradiographic signals obtained after electrophoretic separation of normal cell and AEV cell nuclear RNAs, their transfer to nitrocellulose sheets and hybridization to specific probes. The RNAs in lanes 1 and 2 were hybridized with an aA globin probe, while the RNAs in lanes 3 and 4 were hybridized to an aD probe. In lanes 1 and 3, high mol. wt. (after fractionation on Biogel A50m) nuclear RNA from normal erythroblasts and, in lanes 2 and 4, unfractionated AEV cell nuclear RNA were analysed. Chick ribosomal 18 and 28S rRNA and ASV 35S RNA in parallel lanes served as mol. wt. markers. The autoradiographic signal in lane 1 reveals two distinct populations of molecules containing aA globin mRNA sequences. These correspond to 0.6 kb, the size of mature globin mRNA, and a second larger species of 1.0 kb. Lane 3 shows again the population corresponding to 0.6 kb as in lane 1, and a larger precursor of 1.3 kb for the aD globin. In other words the immediate precursors of the a-globin mRNAs differ in size, the precursor for cxD being larger than that for aA as reported earlier (Therwath and Scherrer, 1982b). AEV cell nuclear RNA, screened in a similar way, failed to give any hybridization signal when probed with the aD cDNA probe (Figure 4, lane 4; see also the dot-blot hybridization in Figure 1). In contrast, there were faint but definite hybridization signals with the aA globin-specific probe. In Figure 4, lane 2, there is a diffuse hybridization signal in a zone corresponding to molecules of 0.6 kb and less. Most notably, a further signal is observed as a discrete band at a position of - 7.3 kb. No trace of any hybridization was detected in the region between these two populations, as observed also in the previous hybridization-titration experiments. Long periods of autoradiographic exposure ( -3 weeks), as well as the relatively high amounts of RNA and labelled cDNA probe necessary to visualize the large but rare globin mRNA specific molecules, resulted in an over exposed image of the abundant cDNA complementary molecules. Short exposures (not shown) indicate molecules of 0.6-0.7 kb for mRNA and 1.0 and 1.3 kb for the pre-mRNA of the aA and aD genes respectively (cf., Therwath and Scherrer, 1982b). The results shown in Figure 4 essentially confirm those in Figure 3. They support the conclusion that in the AEV-transformed chick erythroblasts, an abortive transcription of the globin genes leads eventually to the complete elimination of the globin mRNA within the nucleus. It is important to note that by the techniques used here no signal can be detected in 493
A.Therwath, G.Mengod and K.Scherrer
the 6-12 kb zone in the case of the normal cell nuclear RNA, although other methods have shown globin premRNA in that mol. wt. range (Reynaud, 1980). Discussion We have studied the effect of transformation by an RNA tumor virus, AEV, on the expression of a well studied and differentiation specific family of cellular genes, namely the globin genes, using normal chick erythroblasts as positive controls. A modified dot-blot hybridization technique (Therwath and Strauss, 1981) showed ctA specific gene transcripts present in the AEV-transformed erythroblasts; whereas no products of the aD and ( genes could be detected. However, even this sensitive method would not detect
