Sep 5, 1984 - (Chambon et al., 1979; Gannon et al., 1979; Royal et al.,. 1979 ...... Nunberg,J.H., Kaufman,R.J., Chang,A.C.Y., Cohen,S.N. and Schimke,.
The EMBO Journal vol.3 no.12 pp.2779-2786, 1984
Termination of the ovalbumin
gene
transcription
RNA transcribed in vivo from the chicken ovalbumin gene has been analyzed in detail in the 3' region of the gene using nuclease Si mapping and Northern blotting. We describe two new additional minor polyadenylation sites leading to premessenger RNA with lengths of - 7900 and 9700 nucleotides. Hybridization of RNA transcribed in vitro from oviduct nuclei with various immobilized DNA fragments covering the 3' region of the gene indicates that > 907o of transcription terminates in a discrete region of 170 bp located -900 bp downstream from the 3' end of the last exon. Two copies of a sequence homologous to the one proposed for yeast transcription termination are present within the region where transcription of the ovalbumin gene terminates. Key words: ovalbumin gene/multiple polyadenylation sites/ RNA polymerase B/transcription termination/in vitro nuclear transcription
(Chambon et al., 1979; Gannon et al., 1979; Royal et al., 1979; Heilig et al., 1980,1982a, 1982b; Cochet et al., 1979). We have described the existence of multipe polyadenylation sites for the X and Y genes (Le Meur et al., 1981) and for the ovomucoid gene (Gerlinger et al., 1982). Conflicting results have been published concerning the 3' end of transcripts of the ovalbumin gene. Tsai et al. (1980) and Roop et al. (1980) have mapped the 3' end of the primary transcripts by analyzing RNA purified after in vivo or in vitro nuclear transcription, and have not detected any transcription distal to the major poly(A) site. However, using nuclear transcription in vitro, we have previously found that transcription continues beyond the major poly(A) site of the ovalbumin gene (Gerlinger et al., 1982, and unpublished results). We report here the existence of two functional polyadenylation signals in addition to the major 'classical' one. We also demonstrate that > 900o of transcription terminates in a discrete region located 900 bp downstream from the major polyadenylation site. Based on its homology to a putative yeast termination signal (Henikoff et al., 1983) we propose a sequence which may be involved in the process of transcription termination in the chicken ovalbumin gene.
Introduction The events occurring at the 3' end of RNA polymerase class B (II) transcripts during their maturation have been extensively studied. The hexanucleotide sequence AAUAAA (Proudfoot and Brownlee, 1976) or some variations thereof (see Discussion for references), located 11 -30 nucleotides upstream from the poly(A) tail of eukaryotic mRNAs, functions as a signal for polyadenylation. For some genes there is good evidence that the transcribing RNA polymerases read through poly(A) sites (SV40 late RNAs, adenovirus-2 mRNAs from the major late transcription unit, adenovirus early regions 2 and 4, and the mouse ,B major globin mRNA; Ford and Hsu, 1978; Nevins and Darnell, 1978; Nevins et al., 1980; Fitzgerald and Schenk, 1981; Hofer and Darnell, 1981; Proudfoot, 1984) suggesting that polyadenylation of the mRNAs requires an endonucleolytic cleavage of the primary transcripts followed by poly(A) addition. Transcription termination itself might occur at a considerable distance downstream from the poly(A) site (Hofer et al., 1982). In the case of the H2A histone gene, most transcripts terminate heterogeneously within the 200 bp located downstream from the nucleotide corresponding to the 3' ends of the histone mRNA (Birchmeier et al., 1984; Krieg and Melton, 1984). It thus appears that correct 3' ends of histone mRNA as well as polyadenylated mRNAs are determined by post-transcriptional processing and it is therefore important to determine whether transcription termination occurs progressively or at precise sites in the 3'-flanking region of the genes. The structure and expression of several chicken egg-white genes, i.e., the ovalbumin gene family, the conalbumin and the ovomucoid genes, have been studied in our laboratory
Results Multiple polyadenylated 3' ends of the ovalbumin gene transcripts The map of the ovalbumin gene (Chambon et al., 1979) and of its 3'-flanking sequences is shown in Figure IE and F, respectively. The bold arrow points to the main polyadenylation site of the ovalbumin transcripts. To reveal the possible existence of additional ovalbumin gene transcripts which would not be polyadenylated at the known ovalbumin mRNA poly(A) site, single-stranded DNA fragments (coding strand) covering the ovalbumin gene 3' region were 32P-labelled at either their 3' end (probes a, c and d in Figure IF) or their 5' end (probe b in Figure IF) and hybridized to oviduct poly(A) + or total RNA as well as Escherichia coli RNA as a control. The hybrids were digested with nuclease SI, and the protected DNA fragments analyzed on 7 M urea-polyacrylamide gels. Hybridization of the 867 bp XbaI-Bgll DNA fragment, 3' end-labelled at the XbaI site (probe a in Figure IA, lane 1, and Figure IF), resulted in two protected fragments 527 and 333 nucleotides long (Figure IA, lane 4). The first one corresponds to the classical ovalbumin mRNA, whereas the second minor one reveals the existence of a shorter polyadenylated ovalbumin RNA species. The same two fragments were also found after nuclease SI digestion of hybrids between oviduct polysomal RNA and probe a (not shown). No longer protected DNA fragment could be obtained in this experiment. However when the same XbaI-Bgll DNA fragment was 5' end-labelled at the Bgll site (Figure IF, probe b) and hybridized to oviduct poly(A) + RNA (Figure I B, lane 3), a fraction of the whole probe was protected which suggested that transcription continues beyond the main poly(A) site,
M.A.LeMeur, B.Galliot and P.Gerlinger Laboratoire de Genetique Mol&ulaire des Eucaryotes du CNRS, Unite 184 de Biologie Moleulaire et de Genie Gen&ique de l'INSERM, Faculte de Medicine, 11, rue Humann, 67085 Strasbourg C&dex, France Communicated by P.Chambon
© IRL Press Limited, Oxford, England.
-
2779
M.A.LeMeur, B.Galliot and P.Gerlinger
B
A 1 1631-
C 1 2 3 4 5
1 2 3 4
2 3 4 1118-
go
516
0
& ', -867 525- 4I 516 52 447-7 527 506-*:
,_0
1443-0 1307-354
344-_I 298-_4
X
ai 220-
344-
630-
. -333 Q iGO C
90 0/ of the transcripts in the 170-bp Hael -Hae2 segment, -900 bp downstream from the main polyadenylation site (see map Figure 3C). We have excluded that these results could be due to artefactual in vitro transcription. The synthesized RNA does not correspond to run-through transcription by RNA polymerase molecules present in exon 7, because the same relative hybridization ratio (fragment f versus DNA
Termination of the ovalbunin gene transcrption
fragments lying within the gene) was found irrespective of the time used for incubation of the nuclei (3-30 min). The possibility of random initiation of transcription 3' to the end of exon 7 was investigated by incubating the nuclei in the presence of sarcosyl, heparin and ammonium sulfate (see Materials and methods) which are known to prevent initiation of transcription. Under these conditions, and for a short incubation time (3 min) the transcription pattern was comparable with that obtained in the standard conditions (all the corresponding bands are proportionally less intense, see Figure 3E, lanes 1 and 2). In both cases, there was very little hybridization with fragments Hael-Hae2 and Hae3-HhaI, which strongly suggests that there is no rapid degradation of RNA which may possibly be transcribed from this region, since all known enzymes involved in the RNA processing or degradation should be inhibited by incubating under such conditions. Upon longer incubation (30 min) in the presence of high salt/sarkosyl/heparin, transcription occurred further downstream, since a strong hybridization was obtained with fragment Hhal-Hae4 (compare in Figure 3E, lanes 1 and 2 with lane 3). This observation suggests that some factor involved in transcription termination may be bound to the DNA template when incubation is performed under more physiological conditions. Finally, both the coding and noncoding strands of the XbaI-HindIII fragment (see map Figure 1F) were subcloned in an M13 phage and hybridized with in vitro synthesized nuclear RNA. As expected, hybridization was obtained exclusively with the DNA fragment corresponding to the coding strand (not shown). This excludes the possibility that the hybridization pattern described above could be due to transcription of the non-coding strand of the ovalbumin gene 3'-flanking region.
Discussion We describe here two additional species of polyadenylated ovalbumin transcripts. It is most likely that the first polyadenylation site, which is located 196 bp upstream from the major site (position 7368, Figure 4, and Figure 3B), was not previously observed because nuclease SI mapping of the 3' end of ovalbumin RNA was carried out using a probe labelled 125 nucleotides upstream from the 3' end of exon 7 (Roop et al., 1980). This new polyadenylation site is located 74 bp downstream from an ATTAAA sequence. This exception to the AATAAA sequence is well documented (Nunberg et al., 1980; Jung et al., 1980; Gerlinger et al., 1982). However, such an unusual distance between the consensus signal and the site of polyadenylation has only been reported in the case of the dihydrofolate reductase 1000 nucleotide long RNA (Setzer et al., 1982) and may account for the low efficiency of the polyadenylation process in the case of the ovalbumin gene. It is worth noting that the corresponding polyadenylated RNA was found in polysomes, which suggests that it is translated (result not shown). The great majority of ovalbumin RNA molecules are polyadenylated at the second and 'classical' site (position 7564, Figure 4 and Figure 3B), located 18 bp downstream from an AATAAA sequence. Since we found a small amount of ovalbumin polyadenylated transcripts extending for - 1780 nucleotides past the major polyadenylation site (Figure ID, lane 4 and Figure 2, lane 4), we sequenced the ovalbumin 3'-flanking region down to the Bgl2 site (see map in Figure 3C and D and sequence in Figure 4). A third functional polyadenylation signal, ATTAAA, is indeed located at position 9244 (Figure 4). Multiple poly-
adenylation signals of quite different efficiencies have been reported for other genes (mouse dihydrofolate reductase mRNA, Setzer et al., 1980, 1982; mouse immunoglobulin is and !Lm RNA, Early et al., 1980; mouse a-amylase mRNA, Tosi et al., 1981; bovine prolactin mRNA, Sasavage et al., 1982; mouse immunoglobulin 6 membrane mRNA, Cheng et al., 1982; chicken ovomucoid mRNA, Gerlinger et al., 1982; mouse 32 microglobulin mRNA, Parnes and Robinson, 1983; chicken vimentin mRNA, Zehner and Paterson, 1983; human 3-tubulin, Lee et al., 1983) leading to the suggestion that the use of these multiple polyadenylation sites is correlated with the metabolic state of the cell and therefore has a role in the regulation of gene expression (Nevins and Wilson, 1981; Kaufman and Sharp, 1983). Considering all of the possible polyadenylation signal sequences previously reported, not less than 23 of them can be found within the ovalbumin gene, ATTAAA occurring four times and AATAAA eight times (see also Woo et al., 1981). However only the sequences with the positions referred to above and in Figure 4 have been shown to be used in vivo. We should stress that in the absence of sensitive RNA blotting or nuclease S1 mapping experiments, we do not know whether or not the other possible polyadenylation signal sequences are completely silent. Nuclease S1 mapping, using a single-stranded XbaI-Bgll DNA probe, 5' end-labelled at the Bgll site (Figure IF, probe b), did not reveal any accumulation of RNA molecules cleaved at the polyadenylation site (Figure IB, lane 3). A similar observation has already been reported by Montell et al. (1983) in the case of adenovirus early region IA gene. It appears, therefore, that the transcripts of sequences situated downstream of the polyadenylation site are very unstable when they have been cut for the addition of the poly(A) tail. Thus it is not possible from our results to determine whether polyadenylation occurs before transcription terminates or indeed if the events are linked. No transcripts extending downstream from the third polyadenylation site were detected in total or in poly(A) + RNA, using either nuclease SI mapping or Northern blotting. Therefore nuclear transcription experiments were performed to map the position of RNA polymerase B molecules which are engaged in vivo in ovalbumin gene transcription. Hybridization of oviduct nuclear RNA synthesized in vitro to DNA fragments from the 3'-flanking region of the ovalbumin gene revealed that almost all transcription terminates abruptly within the Hael-Hae2 DNA fragment some 900 bp downstream from the main polyadenylation site (see Figure 3C and E and Results). Preliminary results indicate that transcription terminates also several hundred base pairs downstream from the main polyadenylation site of the conalbumin, ovomucoid, X and Y genes. The termination sites of RNA polymerase B transcription units have not yet been defined, even in the case of the mouse ,B-major globin gene (Hofer et al., 1982; Darnell, 1984). In yeast, the analysis of the effect of precise deletions in the vicinity of the 3' end of the Drosophila ADE-8 gene which was inserted in the yeast 2-nm plasmid, had suggested that the 8-bp TTTTTATA sequence participates in the signal leading to the termination of transcription (Henikoff et al., 1983). Moreover, a 38-bp deletion including this sequence, as well as that of a TATTTATA sequence located a few base pairs downstream in the yeast iso-cytochrome Cl gene (CYCI-512 deletion), has been shown to cause readthrough transcription (Zaret and Sherman, 1982). This 8-bp sequence or derivations thereof are found in several other yeast genes for which the localization of the poly(A) ad2783
M.A.LeMeur, B.Galliot and P.Gerlinger
XbaI V TCTAGAAAAA AAATCAGAAA GAAATTACAC TGTGAGAACA GGTGCAATTC ACTTTTCCTT TACACAGAGT AATACTGGTA ACTCATGGAT GAAGGCTTAA
7037
GGGAATGAAA
7100
TTGGACTCAC AGTACTGAGT CATCACACTG AAAAATGCAA CCTGATACAT CAGCAGAAGG TTTATGGGGG AAAAATGCAG CCTTCCAATT AAGCCAGATA TCTGTATGAC
7200 CAAGCTGCTC CAGAATTAGT CACTCAAAAT CTCTCA*TT AA4ITATCAA CTGTCACCAA CCATTCCTAT GCTGACAAGG CAATTGCTTG TTCTCTGTGT TCCTGATACT
7300 ACAAGGCTCT TCCTGACTTC CTAAAGATGC ATTATAAAAA
TCTT7ATAATT CACATTTCTC CCTAAACTTT GACTCAATCA TGGTATGTTG GCAAATATGG TATATTACTA
7400 TTCAAATTGT TTTCCTTGTA CCCATATGTA ATGGGTCTTG TGAATGTGCT CTTTTGTTCC
7500
TTTAATCA* ATAA*ACAT
GTTTAAGCAA
ACACTTTTCA CTTGTAGTAT
TTGAAGTACA GCAAGGTTGT GTAGCAGGGA AAGAATGACA TGCAGAGGAA TAAGTATGGA .CACACAGGCT AGCAGCGACT GTAGAACAAG TACTAGTGGG TGAGAAGTTG
7600
AACAAGAGTC CCCTACAAGC AACTTAATCT AATAAGCTAG TGGTCTACAT CAGCTAAAAG AGCATAGTGA GGGATGAAAT TGGTTCTCCT TTCTAAGCAT CACCTGGGAC
7700
1-Bglll
7800
. V TCTGTCTCAA AACTCATCTG GAGCAGTGTG TCCAATCTGC CGCTGCCCTG ATCTCGGCTG GGGrGATGGG ACAGACCTTG GCTGCCACTG AGACATCTGA GACACTGAGA
7900 CTCAGATTTA CCCAAGAACA GCTCATTGCC AACAGAACAA AATCTCAAAC TTATGGCTAG TGATGACAGC AGTCAGTTGT CCCATCTGTG ACCCACCAAG GCTGGCATGC
8000 TGGAATGAGC AGGCTTTGGT GGCATGTAGT TACTGGACAG CACCACTGAC ATGGGCAGGG GAAAAACTGA GCATGGTGTA AATCACTGCC TCAAAGCCAC TTCTCTGTGC
Hind III V
CTGCACCATO CTTGAAAGCT CTTCTACAGG AGCTGGGTTT GTTCAAGAAA GCTTCTGTTT CTCCCATCTG CTTCTTGTAC CTTCACAGGG ACAGAGTTAG AAGGGTACAG
Pvu 1
8200
CCATGGCTGG AAGGGGCTGA CTTTCAAATG TGCCTAATTT TCCTTTGGTT GCTGCTGCAG CTGCAGAAGA AGGGGTTCAG AAGCCAAGAG CTTTGAGATA AGGATGCCTA
I -Hae III V
6300
ACCTATGTTG AAGACATTTG TGCTGACACC TCAGGCCCCA GGATAGGACA ACTGCTGGAT TGTGGCTAAC CCACTAGCTA CAGAACCTAA TTTATATTAC CAGATTAGGA
8400
2-Hoe III 3-Hae III V
V
AGAGCAAAAG AACATGTATT TATAACAGGA GGTCTTCTGT GCTTCTCTAC TAAAAGGTGC TGTGAAGGAG CCCACAGTGC AGCAGTGTAT GAGGCCTGAA AGAGGCCGCA
6500
Ava i
GCACACGAAG AGCCCTGGTA GGAGCAGCAC ACAGAGGGGC AGGAGGGCTG GGGGAACTGC CACCCATGGG GACCTGTGTG AAGCAGTGCA CTCCTGAGGG GTGGACTGCG
6600
TGGGAAAGGA AAAGAAAGCA AACAGACCTG TGATGAACTG TCACACAGAC TGCAGAGTGA CAGAGGAGGG CACGAGGCAG TGCGCCCACT GCAGGGAGTG GCGCTCCTTC
8700
CTCACAGCAG CGCTAACAGC TTGGCACCAA TATTCAGTAG TCTGTGGTGA TACTTTTTCC AGTTTCACCA CACAGCATTT CGCTTGTTCT ACTTGTTTTA GCTTTCCCCC
6800
8900
TCCACAAGAT AACACATACT TTGCCAGTCA GTCCCTAAGA CCTTAACTTA ACAGTTAGCA AACAGGATCT TGCAAAAGAA GGAAGATAAC ATGACACCAC CTTCACTGGT
9000
GTATAAATAG TTCAAATACT TTCCTTCACT TTCCCGTAAA TTAGTTGATT GCAGGTCAGG AGATAACAGG GGAACTTACT GCAAGAGAGA AAATGATGTT TAATATTGTC
9100
4-Haelli
TTGGACTTTC TGGTGGTCTG GGCATGAAAA TGGGGTACTC AAAATCCTCG GGACGTTTAT TTTTCACCTG ATTTATTCCC AAACTGCACT ATTTCTAGGC CATTGGAGTT
CTTATCA+rTATACT
CTTATC ATT AA
TTGGCTCTCT
*
9200
TATACT TTGGCTCTCT GCTATCTCAC TCCCTTTCAT CTTCAGCATC ACTTTCAGCA CAATTACAGG AGAAGACTTA GACTCAGAGC TTTAGGACTC
2-BV
III
9300
ATCATAAGAG GCTTTCATTG CTCTGTCACC ACACCCCATA TAGATCT
Fig. 4. Sequence of the 3' end region of the ovalbumin gene (non-coding strand). The strategy followed to sequence the Bgll-Bgt2 DNA fragment, using the Maxam and Gilbert (1980) method, is shown in Figure 3D. Numbering starts at the major cap site of the leader exon. The sequence underlined corresponds to exon 7. The positions of two additional poly(A) sites, as deduced from nuclease SI mapping (Figure 1), are indicated by an asterisk above the sequence. The AATAAA type sequences located upstream from the polyadenylation sites are boxed. Some restriction enzyme sites are indicated, corresponding to those shown in the map of Figure 3C. Sequences a, f and y corresponding to putative transcription termination signals are indicated by arrows (see Discussion).
2784
Termination of the ovalbumin gene transcrption Table I. Sequence homologies between the putative transcription termination signals recognized by yeast polymerase B (plasmid pYFADEB, see Henikoff et al., 1983) and the analogous sequences found in the chicken ovalbumin gene AGAACCT
GAACATG ATCAATT TCAATCT ATAGCAA
TGCG1C
8445 AAT1TATA 8483 TATT[ATA 9247 AAATTATA 5536 AAATTATA 761
FITTlATA 786 T1TTT1ATA
TTACCAGA
arrow a
ACAGGAGG
arrow,B
CTTIGGCTC
arrow y
AAGATAAA
intron G
GG1TTAGGC
pYFADEB plasmid
TAAAAACT
pYFADEB plasmid
Numbers refer to the first nucleotide of the central 8 bp sequences.
dition sites are known (see sequences reported by Zaret and Sherman, 1982) but it has not been unequivocally demonstrated whether the polyadenylated 3' end of the corresponding RNA is due to transcription termination at the polyadenylation site or to processing of the primary transcripts. Analogous sequences are found at two different positions within the ovalbumin gene 3' region where transcription stops (positions 8445 and 8483 in the Hael-Hae2 fragment, see Figure 4 and Table I). A third copy of this sequence is found contiguous to the third polyadenylation signal (position 9247, Figure 4 and Table I). Such sequences seem to be rare, since they are found only once in the entire ovalbumin gene (in intron G, see Table I), twice in the conalbumin gene (10800 nucleotides sequenced, Jeltsch, 1982), seven times in the X gene (10720 nucleotides sequenced, Heilig, personal communication), and do not exist in the Y gene (8730 nucleotides sequenced, Heilig et al., 1982b), which suggests that the T l-llTTTATA sequence or derivative thereof may be necessary, but not sufficient, for transcription termination. Whether or not additional upstream or downstream sequences or secondary DNA or RNA structures could also be implicated is unknown. A computer analysis revealed several palindromic structures and possible Z-DNA configuration within the 3'-flanking region of the ovalbumin gene. However, such structures are present at the same frequency within the ovalbumin gene itself and their significance, if any, remains to be assessed. However, that RNA polymerase molecules read through the termination regions when nuclei were incubated in the presence of sarkosyl/heparin/high salt, suggests that some factor(s) bound to the DNA template may be involved in termination of transcription. We are currently investigating whether the putative terminator region of the ovalbumin gene, inserted in chimeric recombinants, can cause termination of transcription after transfer of the recombinants into a variety of cells in culture. Materials and methods RNA preparation Oviduct total RNA was prepared by the method of Auffray and Rougeon (1980) with minor modifications (LeMeur et al., 1981). Polyadenylated RNA was isolated by affinity chromatography on oligo(dT)-cellulose column (Aviv and Leder, 1972). Gel electrophoresis of RNA, transfer to DBM-paper and hybridization to nick-translated DNA probes was carried out as described earlier (LeMeur et al., 1981; Gerlinger et al., 1982).
Nuclease SI mapping The coding strand of DNA restriction fragments was labelled at either the 5'
end with polynucleotide kinase and [-y-32P]ATP or the 3' end with DNA polymerase I and [a-32P]deoxynucleotide triphosphates (see Figure IF). The strands were separated by polyacrylamide gel electrophoresis (Maxam and Gilbert, 1980). A total of 5-50 ng of fragments were hybridized to 21000 jig of RNA and treated with nuclease S1 under the conditions described by Heilig et al. (1980). The size of the nuclease SI resistant fragments was determined by electrophoresis on 3-5 % polyacrylamide sequencing gels. Preparation of nuclei, transcription and isolation of RNA for hybridization Nuclei were prepared as described by Gariglio et al. (1980), adjusted to 1.5 mg DNA/ml, frozen in liquid nitrogen and stored in aliquots at - 80°C. Transcription was performed at 25°C for 45 min in 100 A1 reactions containing 40 Al nuclei, 1.2 mM ATP, GTP and UTP, 100 ACi [a-32P]CTP (sp. act. 400 Ci/mmol) and either 12 mM Tris-HCl pH 8, 120 mM KCI and 5 mM MgCl2 (standard conditions) or 87 mM Tris-HCl, pH 8, 24 mM NaCI, 0.2 mM EDTA, 10 mM MgCl2, 350 mM ammonium sulfate, 1 mg/ml heparin and sarkosyl 0.6% (sarkosyl/heparin/high salt conditions). RNA was extracted from nuclei as in McKnight and Palmiter (1979). Filter hybridization Ovalbumin-specific DNA fragments were either denatured and immobilized on nitrocellulose filters or separated by 307o low melting preparative (LMP) agarose slab gel in Tris-acetate-EDTA buffer (G.Dretzen, personal communication), and transferred to DBM-paper (Wahl et al., 1979). Conditions for hybridization and subsequent treatment of the filters were as described by Hofer and Darnell (1981).
Acknowledgements We are indebted to Professor P.Chambon for continued interest in the work and for useful discussions. We thank D.Duboule and G.Richards for critical readings of the manuscript. The excellent technical help of P.Hickel is gratefully appreciated. We thank P.Meyer and E.Taubert for technical assistance, C.Werle and B.Boulay for preparing the figures, C.Kutschis and C.Aron for typing the manuscript. This work was supported by grants from the CNRS (ATP 6182) the INSERM (PRC 118012), the Fondation pour la Recherche Medicale, the Association pour le Developpement de la Recherche sur le Cancer and the Fondation Simone and Cino del Duca.
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