BIOCHEMICAL
Vol. 184, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
April 30, 1992
945-952
HETEROGENEITY a 3’UNTRANSLATED REGION m BOVINE ACIDIC FGFTRANSCRIPTS Flare Renaud, Sophie Desset, Ku!-as Bugra, CXcile Halley, Jean-Marc Philippe,Yves Courtois and Rlaryyonne Laurent Unite de Recherches Gerontologiques, INSER!! U118, afftliee CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France
Received
February
7,
1992
A bovine aFGF genomic clone (14.2Kb) has been isolated and characterized. This clone contains exons 2 and 3 interrupted by 6.7 Kb long intron. Exon 3 contains part of the coding region and the 3’ untranslated region (3’UTR). Two overlapping cDNA clones specific for this 3’UTR have been isolated from bovine retina cDNA libraries or after amplification of RNA by the RACE technique. Analysis of these clones and RNAse protection assay demonsnate alternative termination of aFGF transcripts giving rise to differently sized 3’UTR of 2.5 Kb and at least 3.9 Kb. The sequence of these long 3’UTR is highly conserved among species(70% identity between human and rat) which suggests an important role for aFGF expression. 0 1992 Academic Press, Inc.
Acidic fibroblast growth factor is a member of the heparin binding protein family including growth factors such as basic fibroblast growth factor ( bFGF) (1) , keratinocyte growth factor (KGF) (2) and oncogene products: int-2 (3), hst/kFGF (4,5), FGF5 (6). FGF6 (7) and the recently identified genes MK (S), RIHB (9), HBNF (10). The members of the FGF subfamily ( a and b FGF, KGF, int2, hst, FGFS, FGF6) share significant amino acid sequence homology and the coding regions of these genes are interrupted by 2 introns where the exon-intron junctions are conserved. The untranslated regions (UTR) show no homology. cDNAs encoding acidic FGF have been isolated in human (1 l), bovine (13,14), rat (15), chicken (16) and pig (17). In these different species, the nucleotide sequences of the aFGF coding regions show more man 85% identity. The 3’ UTR in bovine, chick and pig are only partially characterized; in rat and human, 3’UTR are 44s bp and 3119 bp and share 70% sequence identity. The 3‘UTR in human contains multiple polyadenylation sites which partially explain the heterogeneity of the human aFGF transcripts (18,12). We previously reported the sequence of the 2546 bp of the bovine 3’UTR (14). That sequence, which lacked poly A tail and polyadenylation sites, was 70% homologous to the human 3’UTR up to the position 2755 where the two sequencestotally diverged. This bovine aFGF cDNA clone hybridized to different size mRNAs in brain and retina, the major ones being a 4.2 Kb mRNA coding for aFGF and a 2.5 Kb mRNA which specifically hybridized the 3’ end of the 3’UTR (13). Since the 3’UT region of aFGF gene is long and well conserved in the different species,we speculated that it could be involved in the control of the level of aFGF transcripts. Therefore we have completed the characterization of the bovine 3’UTR and elucidated on the 2.5 Kb mRNA. To this end, additional cDNA and genomic clones have been isolated and characterized. Here we report that several polyadenylation sites differently localized in human and bovine aFGF genes are used for transcription of the multiple aFGF
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mRNAs. A new minor transcript with a 3’UTR at least 3.9 Kb has also been characterized in bovine brain and retina. hlETHODS Northern analvsis:Total RNA was extracted from bovine retina using the guanidium isothiocyanate procedure(19).oly A+ RNA was further purified on oligo dT cellulose affinity column and electrophoresed on a formaldehyde agarose gel (20). RNAs were blotted on Hybond N+ membrane and hybridized with (3’P) random primed probes. Screening --A of the libraries.Two bovine retina cDNA libraries were screened using different fragments of the KB5 bovine aFGF cDNA clone (13). Additional cDNA clones were synthetized using the RACE ( Rapid Amplification of cDNA Ends) protocole (21). Briefly lug of total RNA from bovine retina was reverse transcribed using an oligodT.ClaI adaptater primer. Single stranded cDNAs were then amplified between this oligo dT adaptater primer and an aFGF specific primer linked to the ClaI adaptater. The aFGF specific sequence was located at positions 2395-2415 from the ATG. The amplified DNA was restricted with ClaI and KpnI and subcloned in the bluescript vector. A bovine genomic DNA library constructed by partial Hind111 digestion of thymus DNA was kindly provided by Transgene (Strasbourg). A second genomic library constructed by inserting partial MboI digested adult bovine DNA in the EMBL3 vector was also screened (Clontech). One million clones from each library were screened with CaP)labelled aFGF cDNA probes. DNA seouencins analvsis:The nucleotidique sequences of the cDNA and genomic fragments subcloned in the bluescript vector were done on alkali denaturated DNA purified on a sephactyl S400 column using the T7 dideoxy sequencing kit (Pharmacia LKB). The sequences were analyzed using citi2 BISANCE sequence databases(22). RNAseprotection assa=The cDNA clone 471 and the KpnI-Pstl fragment of the genomic clone FR2.1 were inserted in the bluescript vector, Linearized DNA was used as a template for the T7 or T3 RNA polymerases to produce uniformly labelled antisens RNA probes. 2 pg of poly A+ RNA from bovine brain and retina were hybridized to (3’P) labelled RNA probe (lo7 cpm). The hybridization and RNAse treatment were done according to the ribonuclease protection assay kit from Ambion INC, except that the hybridization temperature was 48°C. The protected tiagments were electrophoresed on a 6% denaturating acrylamide gel. The sizes of the protected fragments were determined by using Sau3A and Mae111bluescript fragments labelled with the polynucleotide kinase. Nuclmtide sequences of the same region was also used as markers.
KESUJ,TS 1.RNA ANALYSIS By NORTHERN HYBRIDIZATION 10 kig of poly A+ RNA from bovine retina was hybridized with various fragments covering the coding region and the 3’UTR of the bovine aFGF cDNA clone KB5 (Fig 1). Up to the KpnI site, all the probes hybrized the 9.9 Kb and 4.2 Kb transcipts. The Kpnl-SmaI fragment hybridized the 2.5 and 4.2 Kb mRNAs with the same intensity, while the SmaI-EcoRl fragment only hybridyzed the 2.5 Kb transcript. The analysis of the nucleotide sequence in that region revealed an open reading frame coding for 119 amino acids which starts with an ATG at position 3572 and ends at position 3929. 2. ANALYSIS OF THE cDNA CLONES. The cDNA libraries from bovine retina were screened with the KpnI-Smai and the SmaIEcoRI fragments of the clone KB5. Several clones which specifically hybridized to both probes were selected: 2 only hybridized to the KpnI-SmaI probe and 10 hybridized to the Smal-EcoRI probe. Using the RACE protocole a 600 bp cDNA clone (M4) was also isolated and characterized. Only the KpnI-SmaI specific clones cross-hybridized with the M4 probe. The longest one: a 918 bp long clone (471) was further analyzed, The partial restriction map of !v14and 471 clones were established and the 946
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cONA CLONE KB5 EcoRl i
BamHl
Sphl
Sphl
Ndel
Kpnl
4
JI
JI
+
$A
+ ORFl + ATG TAA
2 -
non coding coding region
H
100 bp
I
Smal EcoRl
+ORF,+ ATG TGA
3
4
5
9.9
rn
4.2
2.5
6
Fip. 1. Northern blottine analysis. A. 10 pg of poly A+ mRNA derived from bovine retina was hybridized with full length bovine aFGF cDNA clone KB5 (1) and with different restriction frao_ments of this clone: BarnHI-Suhl (2). Sohl’ ’ SphI (3), NdeILKp61’(4), KpnI-SmaI (5), Smal-EcoRI j6). RNA sizes are given in Kb.’ B. The different fragments used for Northern blot analysis are indicated with arrows. The open boxes represent the coding region: ORFl coding for aFGF, ORF2 coding region deduced from the nucleotide sequence.
clones were mapped to the KB5 clone (Fig 2). The nucleotide sequences of the clones 471 and M4 were 100% homologous to the sequence of the clone KB5 but totally diverged at a position 2755 inside the second open reading frame where the human and the bovine 3’UTR sequences also diverged. The clone M4 had a poly A tail and a classical polyadenylation site, while the clone 471 extended further to the 3’ end and had no poly A tail. 3, ANALYSIS -__ OF THE BOVINE GENOMIC aFGF CLONE. A partial restriction map of the 14.2 Kb insert of the FR2.1 genomic clone was established and various fragments were subcloned into the BS(-) vector (Fig3). Southern blotting analysis established that the 3 fragments 4.1 Kb Kpnl-EcoRI, 5.1 Kb EcoRI-KpnI and 2.5 Kb KpnI-EcoRI, contained 2 exons of &he aFGF gene. Exon 2 of the coding region was located in the 4.1 Kb KpnI-EcoRI fragment. It was 104 bp long and was separated from the exon 3 by a 6.7 Kb intron. The inn-on at the 5’ end of exon 2 was incomplete (22.8 Kb). Exon 3 was entirely contained in the EcoRI-KpnI (5.1 Kb) and the KpnI-EcoRI (2.5 Kb) fragments and was completely sequenced (Fig.4). Sequence analysis showed 100% homology with the KB5 cDNA clone until position 2755. Beyond that nucleotide the sequence was 100% homologous to the clone 471. As in the human, the long bovine exon 3 was unspliced. The sequencesof the 3’ and 5’ intron-exon boundaries matched the classical acceptor/donor splice site sequencesand were located at exactly the same positions as human
!a!5
I E
ORFl
-
non coding
t-l
100 bp
I
2577'
3492
coding region
E :EcoRI
K:Kpnl
C :Clal
Fie.2. Map of three overlaouing ______ aFGF cDNA clones: KB5.471 and M4. The two clones KB5 and 471 were isolated after the screening of bovine retina cDNA libraries. The clone M4 was isolated usin,g the RACE method, The boxes indicate open reading frames as in Fig. 1. (A)n indicates the poly A tad of the clone M4.
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EcoRl
Kpnl
EcoRl
Kpnl
Ed4
4
4
+
4 EXON 3
+
EXON 2 1wbp
EMBLJ
EMBL3 pm
INTRON >2,8 Kb
INTRON
lkb -
6.7 Kb
I
coding region
0
non coding region
-
mtron
Fig.3. Partial restriction map of aFGF penomic clone FR2.1 isolated from a partial Mbol bovine genomic DNA library screened with aFGF cDNA clone KB5. The clone contained exon 2 and 3, part of intron 2 and the complete intron 3.
Gin Ix" Gin mu cys Ala GlU .Ser Ile cay Tr CAG CTG CAG CTC TGT GCG ma AGC ATA GGG
ttttgctcgtgttatttttatttcag
Glu "al Tyr Ile Lys Ser Thr Glu Thr Gly Gln Phe Leu Ala Met Asp Thr Asp GAG GTG TAT AT-7 MG AGT ACG GAG ACT CGC CAG TTC TTG GCC ATG GAC ACC GAC
202 256
Gin Thr pi-0
~cgaaadCCaCtCaCtaattattctactctc:ctggttttattattttttttag CXG ACA ccc
283
Asn Glu c-1" Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr AAT GAG GAA TGT T-E TTC CTG GAA AGG TTG GAG GAA MC CAT TX
Asn Thr ~yr AX ACC TAC
317
Ile Ser Lys Lys His Ala Glu Lys His Trp Phe "al Gly Leu Lys Lys As" sly ATX TCC A&G MG CAT GCA GAG AAG CAT TGG TTC GTT GGT CTC AAG AAG .&AC GGA
391
Ar¶ Ser LYS Leu Gly Pro Arg Thr His Phe Gly Gln Lys Ala Ile Leu Phe LOU AGG TCT AM CTC GGT CCT CGG ACT CAC TTC GGC CAG AAA GCC ATc TTG TIT CTC
445
Pro Leu Pro v.31 Sex ser
Asp
RGGMGiCC~GGATGATGTCXGtTGGTGGAACTGGGG
1230
CMAGTPCATTTGGGTCTGGGGAGTGGGAGTGATA -
1102
~CAGTGGGTCTGTGGTGGAG~CCCAITTCTTACAACC
1518
kCTCCACCCCC~CACATTI\AATAGCXAGGTCCCTAXZACCC~GA CCCCATGCCCCATGGATCTTCCTCCAC'PCAGGCAGTPA
1590 1662
Fig.4.Nucleotide and amino acid seauences of bovine aFGF penomic clone FR2.1.The clone was sequenced by the chain termination method with double stranded DNmen%%ed by alkaline procedures. The first nucleotide corresponds to the ATG (Halley et al. 1989). The consensus polyadenylation signal sequences AATAAA are boxed. The poly A stretch is indicated with a wavy line. The 3’ destabilization motifs ATTT and TATT are underlined. 948
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The sequence of the 3’ UTR showed 36 ATTT and 23 TATT motifs and several classicaland unusual polyadenylation sites (23). The two classicalones are located at positions 2837 and 2986; the last one followed by an internal genomic poiy A strech. To determine which of these sites are used by the 471 and M4 clones, an RNAse protection assaywas performed using different fragments of the 3’ UTR (FigS). RNA probes containing the 2 classical polyadenylation sites were produced by transcription of the clone 471 and hybridized with poly A+ RNA from bovine brain and retina. One major protected fragment of 420 bp corresponding to the AATAAA at position 2986 was protected by both RNAs. Thus the M4 clone corresponds to the major aFGF transcript. A faint band corresponding to the full length probe was also visible. Additional RNAse protection assays showed that the RNA probe SspI-Pstl was also completely protected suggesting that the clone 471 corresponds to a minor aFGF transcript ending further downstream from the PstI site. 949
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2345
-
&
*
3’ END OF FR2.1
GENOMIC
?
a
e
420 340
CLONE)
-
RNA antisens probe protected fragment
Fig.5. RNAseurotectionassav The cDNA clone 471 (2577-3492) was subcloned in bluescriut KS(-). After linearization at the 5’ end, the antisense probe corresponding to this clone was produced by T7 RNA polymerase (BRL) in presence of (32P)UTP (Amersham). The restriction fragment KpnI-Pstl from genomic clone FR2.1 was subcloned in bluescript KS(-). After linearization by SspI (4098), antisense RNA corresponding to the PstI-SspI (4098-4378) fragment was produced. 2 ug of polyA+ mRNA isolated from bovine retina (lanes 2,3) and brain (lanes 4,s) were hybridized at 48°C with IO7 cpm antisense RNA 471 (3,5) and antisense RNA SspI-Pstl (2,4). Digestion with Tl and A RNAse was performed using an RNAse protection assay kit (ORION). The sizes of the protected fragments were determined using double stranded DNA sequencing of KnpI-PstI genomic subclones with specific synthetic oligonucleotides (lane 1, GATC). The arrows indicate the sizes of Mae111 and Sau3A bluescript restriction fragments used as markers.
DISCUSSION In the bovine the most highly expressed aFGF transcript is 4.2 kb, minor mRNAs of 1 kb and 9.9 kb are also detected in bovine brain and retina. In that species a 2.5 kb mRNA encoded by the 3’ end of the untranslated region was also expressed in these two tissues (13). The characterisation of the genomic and several cDNA clones shows that the 2.5 kb message is not encoded by the aFGF gene. We do not know the identity of this transcript that is expressed both in the brain and retina.The open reading frame which hybridized to it, is a result of a cloning artefact occurring inside the ORF 2 during the construction of the library. We observed such events in commercial and laboratory prepared libraries during the isolation of different genes. This fact implies that, specially for transcripts which have unusual long untranslated regions. several cDNAs from different speciesshould be analyzed and compared. In bovine, the coding region of bovine aFGF is interrupted by two large introns leading to a 101 bp middle exon. The exon 2 has the same size and the same 3’ and 5’ inuon-exon junctions as the other members of the aFGF family. The 3’ UTR of the 4.2 kb m&VA is 2.5 kb impliing a 1.2 kb 5’
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UTR. Such long UTR are unusual and probably involved in control of transcription of the aFGF gene. The classical poly adenylation site AATAAA used in the 4.2 kb mRNA is immediately followed by a polyA stretch which is not found in the human gene. Downstream from this polyA site, the terminator consensus sequencesnecessary for the cleavage of the transcripts (23) are not present, while such sequences are used in human to generate three minor transcripts at minor consensus polyadenylation sites (13). A minor transcript, corresponding to the clone 471, has a 3’UTR of at least 3.9 kb since the corresponding polyadenylation signal has not been identified in the genomic clone. This 3’UTR is the longest one actually reported for aFGF. The steady state levels of aFGF is very low which may result from a low level of transcription and/or from instability of the message. In addition to the multiple polyadenylation sites the 3’UTR also contains potential destabilization elements, As in human the motif ATITA is repeated 6 times within 780 bp of the 3’UTR. Three of them are located at the same positions in human and bovine species. 32 ATTT motifs are also present in the bovine 3’UTR; this sequence is repeated 24 times in human and only 2 times in the rat. These sequenceshave been shown to destabilize lymphokines, cytokines and proto-oncogene mRNAs (24) and may be involved in controlling the steady state levels of aFGF mRNA (25). On the other hand the role of 3’ UTR in controlling the transcription of one member of the FGF family (hst) has been established. Such mechanisms could also be used for the aFGF gene. Experiments are currently in progress to test this hypothesis.
ACKNOmEDGMENTS
We are grateful to He& Coet for photographic assistance, to Alain HaslC for drawings and David Hicks for hepful discussions. This work was supported by fellowships from Ministere de la Rechercheet de la Technologie and from the Fondation IFSEN.
REFERENCES l- Abraham J.A., Whang J.L., Tumolo A., Mergia A., Friedman J.. Gospodarowics D., Fiddes J.C. (1986) EMBO J. 5,2523-2528 2- Finch P.W., Rubin J.S., rMiki T., Ron D., Aaronson S.A. (1989) Science 245, 752-755. 3- Dckson C., Peters G. (1987) Nature 326. 833 4- YoshidaT., Miyagawa‘K., ddagiri H., Sakamoto H., Little P.F.R.,Terada M., Sugimura T. (1987) Proc. Natl. Acad. Sci. USA 84, 7305-7309 5 Delli-Bovi P., Curatola A.M., Newman K.M., Sato Y.. Moscatelli D., Hewick D., Ritkin D.B., Basilic0 C. (1988) Mol. Cell. Biol. 8, 2933-2941 6- Zhan X., Bates B., Ku X., Goldfarb M. (1988) Mol. Cell. Biol. 8, 3487-3495 7- Marics I., Adelaide J., Raybaud F., Mattei M.G., Coulier F., Planche J.. de Lapeyriere 0.. Bimbaum D. (1989) Oncogene 4.335340 8-Matsubara S., Tomomura IM., Kadomatsu K., Muramatsu T. (1990) J. Biol. Chem. 265,9441-9443 9-Urios P., Duprez D., Le Caer J.P., Courtois Y, Vigny M., Laurent M. (1991) Biochern. Biophys. Res. Comm. 175.617-624 lo-Merenmies J., Rauvala H. (1990) J. Biol. Chem. 265, 16721-16724 1l- Jaye M;, Howk R., Burgess W., Ricca G.A., Chiu I.M., Ravera M.W., O’Brien S.J., Modi W.S., Maciag T., Droman W.N. (1986) Science 233,541-544 12- Crumley G.R., Howk R.. Ravera M.W., Jaye M. (1989) Gene 85, 489-497. 13- Alterio J., Halley C., Brou C., Soussi T., Courtois Y, Laurent XI. (1988) FEBS Lett. 242,41-46 14- Halley C., Courtois Y, Laurent M. (1988) Nucl. Acid. Res. 16. 10913 15- Goodrich S.P., Yan G.C., Bahrenburg K., Mansson P.E. (1989) Nucl. Acid. Res. 17, 2867 16- Schnurch H., Risau W. (1991) Development 111, 1143-1153 17- Schmidt h?;, Sharma H.S., Schott R.J., Schaper W. (1991) Biochem. Biophys. Res. Comm. 180, 853-859 951
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18- Chiu I.M., Wang W.P., Lehtoma K. (1990) Oncogene 5, 755-762 19- Chirgwin J.M., Przybyla A.E., MC Donald R.J., Rutter W.J. (1979) Biochemistry 18, 5294. 5299 20-Innis M.A, Gelfand D.H., Sninsky J.J., White T.J. (1990) PCR protocols: a guide to methods and applications Acad. press INC 21- Man&is T., Fritsch E.F., Sambrook J. (ed.) (1982) Molecular cloning : a laboratory manual Cold Spring Harbor Laboratory; Cold Spring Harbor New-York 22- Dessen P., Fondrat C., Valencien C., 1Mugnier C. (1990) CABlOS 6,355356 23.Birstiel M.L., Busslinger M.,Strub K. (1985) Cell 41.349-359 24.Reeves R., Elton T.S., Nissen M.S., Lehn D., Johnson K.R. (1987) Proc. Natl. Acad. Sci. USA 84,6531-6535 25Curatola A.M., Basilic0 C. (1990) Mol. Cell. Biol. 10,2475-2484
952
Vol. 184, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
April 30, 1992
945-952
HETEROGENEITY a 3’UNTRANSLATED REGION m BOVINE ACIDIC FGFTRANSCRIPTS Flare Renaud, Sophie Desset, Ku!-as Bugra, CXcile Halley, Jean-Marc Philippe,Yves Courtois and Rlaryyonne Laurent Unite de Recherches Gerontologiques, INSER!! U118, afftliee CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France
Received
February
7,
1992
A bovine aFGF genomic clone (14.2Kb) has been isolated and characterized. This clone contains exons 2 and 3 interrupted by 6.7 Kb long intron. Exon 3 contains part of the coding region and the 3’ untranslated region (3’UTR). Two overlapping cDNA clones specific for this 3’UTR have been isolated from bovine retina cDNA libraries or after amplification of RNA by the RACE technique. Analysis of these clones and RNAse protection assay demonsnate alternative termination of aFGF transcripts giving rise to differently sized 3’UTR of 2.5 Kb and at least 3.9 Kb. The sequence of these long 3’UTR is highly conserved among species(70% identity between human and rat) which suggests an important role for aFGF expression. 0 1992 Academic Press, Inc.
Acidic fibroblast growth factor is a member of the heparin binding protein family including growth factors such as basic fibroblast growth factor ( bFGF) (1) , keratinocyte growth factor (KGF) (2) and oncogene products: int-2 (3), hst/kFGF (4,5), FGF5 (6). FGF6 (7) and the recently identified genes MK (S), RIHB (9), HBNF (10). The members of the FGF subfamily ( a and b FGF, KGF, int2, hst, FGFS, FGF6) share significant amino acid sequence homology and the coding regions of these genes are interrupted by 2 introns where the exon-intron junctions are conserved. The untranslated regions (UTR) show no homology. cDNAs encoding acidic FGF have been isolated in human (1 l), bovine (13,14), rat (15), chicken (16) and pig (17). In these different species, the nucleotide sequences of the aFGF coding regions show more man 85% identity. The 3’ UTR in bovine, chick and pig are only partially characterized; in rat and human, 3’UTR are 44s bp and 3119 bp and share 70% sequence identity. The 3‘UTR in human contains multiple polyadenylation sites which partially explain the heterogeneity of the human aFGF transcripts (18,12). We previously reported the sequence of the 2546 bp of the bovine 3’UTR (14). That sequence, which lacked poly A tail and polyadenylation sites, was 70% homologous to the human 3’UTR up to the position 2755 where the two sequencestotally diverged. This bovine aFGF cDNA clone hybridized to different size mRNAs in brain and retina, the major ones being a 4.2 Kb mRNA coding for aFGF and a 2.5 Kb mRNA which specifically hybridized the 3’ end of the 3’UTR (13). Since the 3’UT region of aFGF gene is long and well conserved in the different species,we speculated that it could be involved in the control of the level of aFGF transcripts. Therefore we have completed the characterization of the bovine 3’UTR and elucidated on the 2.5 Kb mRNA. To this end, additional cDNA and genomic clones have been isolated and characterized. Here we report that several polyadenylation sites differently localized in human and bovine aFGF genes are used for transcription of the multiple aFGF
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mRNAs. A new minor transcript with a 3’UTR at least 3.9 Kb has also been characterized in bovine brain and retina. hlETHODS Northern analvsis:Total RNA was extracted from bovine retina using the guanidium isothiocyanate procedure(19).oly A+ RNA was further purified on oligo dT cellulose affinity column and electrophoresed on a formaldehyde agarose gel (20). RNAs were blotted on Hybond N+ membrane and hybridized with (3’P) random primed probes. Screening --A of the libraries.Two bovine retina cDNA libraries were screened using different fragments of the KB5 bovine aFGF cDNA clone (13). Additional cDNA clones were synthetized using the RACE ( Rapid Amplification of cDNA Ends) protocole (21). Briefly lug of total RNA from bovine retina was reverse transcribed using an oligodT.ClaI adaptater primer. Single stranded cDNAs were then amplified between this oligo dT adaptater primer and an aFGF specific primer linked to the ClaI adaptater. The aFGF specific sequence was located at positions 2395-2415 from the ATG. The amplified DNA was restricted with ClaI and KpnI and subcloned in the bluescript vector. A bovine genomic DNA library constructed by partial Hind111 digestion of thymus DNA was kindly provided by Transgene (Strasbourg). A second genomic library constructed by inserting partial MboI digested adult bovine DNA in the EMBL3 vector was also screened (Clontech). One million clones from each library were screened with CaP)labelled aFGF cDNA probes. DNA seouencins analvsis:The nucleotidique sequences of the cDNA and genomic fragments subcloned in the bluescript vector were done on alkali denaturated DNA purified on a sephactyl S400 column using the T7 dideoxy sequencing kit (Pharmacia LKB). The sequences were analyzed using citi2 BISANCE sequence databases(22). RNAseprotection assa=The cDNA clone 471 and the KpnI-Pstl fragment of the genomic clone FR2.1 were inserted in the bluescript vector, Linearized DNA was used as a template for the T7 or T3 RNA polymerases to produce uniformly labelled antisens RNA probes. 2 pg of poly A+ RNA from bovine brain and retina were hybridized to (3’P) labelled RNA probe (lo7 cpm). The hybridization and RNAse treatment were done according to the ribonuclease protection assay kit from Ambion INC, except that the hybridization temperature was 48°C. The protected tiagments were electrophoresed on a 6% denaturating acrylamide gel. The sizes of the protected fragments were determined by using Sau3A and Mae111bluescript fragments labelled with the polynucleotide kinase. Nuclmtide sequences of the same region was also used as markers.
KESUJ,TS 1.RNA ANALYSIS By NORTHERN HYBRIDIZATION 10 kig of poly A+ RNA from bovine retina was hybridized with various fragments covering the coding region and the 3’UTR of the bovine aFGF cDNA clone KB5 (Fig 1). Up to the KpnI site, all the probes hybrized the 9.9 Kb and 4.2 Kb transcipts. The Kpnl-SmaI fragment hybridized the 2.5 and 4.2 Kb mRNAs with the same intensity, while the SmaI-EcoRl fragment only hybridyzed the 2.5 Kb transcript. The analysis of the nucleotide sequence in that region revealed an open reading frame coding for 119 amino acids which starts with an ATG at position 3572 and ends at position 3929. 2. ANALYSIS OF THE cDNA CLONES. The cDNA libraries from bovine retina were screened with the KpnI-Smai and the SmaIEcoRI fragments of the clone KB5. Several clones which specifically hybridized to both probes were selected: 2 only hybridized to the KpnI-SmaI probe and 10 hybridized to the Smal-EcoRI probe. Using the RACE protocole a 600 bp cDNA clone (M4) was also isolated and characterized. Only the KpnI-SmaI specific clones cross-hybridized with the M4 probe. The longest one: a 918 bp long clone (471) was further analyzed, The partial restriction map of !v14and 471 clones were established and the 946
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cONA CLONE KB5 EcoRl i
BamHl
Sphl
Sphl
Ndel
Kpnl
4
JI
JI
+
$A
+ ORFl + ATG TAA
2 -
non coding coding region
H
100 bp
I
Smal EcoRl
+ORF,+ ATG TGA
3
4
5
9.9
rn
4.2
2.5
6
Fip. 1. Northern blottine analysis. A. 10 pg of poly A+ mRNA derived from bovine retina was hybridized with full length bovine aFGF cDNA clone KB5 (1) and with different restriction frao_ments of this clone: BarnHI-Suhl (2). Sohl’ ’ SphI (3), NdeILKp61’(4), KpnI-SmaI (5), Smal-EcoRI j6). RNA sizes are given in Kb.’ B. The different fragments used for Northern blot analysis are indicated with arrows. The open boxes represent the coding region: ORFl coding for aFGF, ORF2 coding region deduced from the nucleotide sequence.
clones were mapped to the KB5 clone (Fig 2). The nucleotide sequences of the clones 471 and M4 were 100% homologous to the sequence of the clone KB5 but totally diverged at a position 2755 inside the second open reading frame where the human and the bovine 3’UTR sequences also diverged. The clone M4 had a poly A tail and a classical polyadenylation site, while the clone 471 extended further to the 3’ end and had no poly A tail. 3, ANALYSIS -__ OF THE BOVINE GENOMIC aFGF CLONE. A partial restriction map of the 14.2 Kb insert of the FR2.1 genomic clone was established and various fragments were subcloned into the BS(-) vector (Fig3). Southern blotting analysis established that the 3 fragments 4.1 Kb Kpnl-EcoRI, 5.1 Kb EcoRI-KpnI and 2.5 Kb KpnI-EcoRI, contained 2 exons of &he aFGF gene. Exon 2 of the coding region was located in the 4.1 Kb KpnI-EcoRI fragment. It was 104 bp long and was separated from the exon 3 by a 6.7 Kb intron. The inn-on at the 5’ end of exon 2 was incomplete (22.8 Kb). Exon 3 was entirely contained in the EcoRI-KpnI (5.1 Kb) and the KpnI-EcoRI (2.5 Kb) fragments and was completely sequenced (Fig.4). Sequence analysis showed 100% homology with the KB5 cDNA clone until position 2755. Beyond that nucleotide the sequence was 100% homologous to the clone 471. As in the human, the long bovine exon 3 was unspliced. The sequencesof the 3’ and 5’ intron-exon boundaries matched the classical acceptor/donor splice site sequencesand were located at exactly the same positions as human
!a!5
I E
ORFl
-
non coding
t-l
100 bp
I
2577'
3492
coding region
E :EcoRI
K:Kpnl
C :Clal
Fie.2. Map of three overlaouing ______ aFGF cDNA clones: KB5.471 and M4. The two clones KB5 and 471 were isolated after the screening of bovine retina cDNA libraries. The clone M4 was isolated usin,g the RACE method, The boxes indicate open reading frames as in Fig. 1. (A)n indicates the poly A tad of the clone M4.
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EcoRl
Kpnl
EcoRl
Kpnl
Ed4
4
4
+
4 EXON 3
+
EXON 2 1wbp
EMBLJ
EMBL3 pm
INTRON >2,8 Kb
INTRON
lkb -
6.7 Kb
I
coding region
0
non coding region
-
mtron
Fig.3. Partial restriction map of aFGF penomic clone FR2.1 isolated from a partial Mbol bovine genomic DNA library screened with aFGF cDNA clone KB5. The clone contained exon 2 and 3, part of intron 2 and the complete intron 3.
Gin Ix" Gin mu cys Ala GlU .Ser Ile cay Tr CAG CTG CAG CTC TGT GCG ma AGC ATA GGG
ttttgctcgtgttatttttatttcag
Glu "al Tyr Ile Lys Ser Thr Glu Thr Gly Gln Phe Leu Ala Met Asp Thr Asp GAG GTG TAT AT-7 MG AGT ACG GAG ACT CGC CAG TTC TTG GCC ATG GAC ACC GAC
202 256
Gin Thr pi-0
~cgaaadCCaCtCaCtaattattctactctc:ctggttttattattttttttag CXG ACA ccc
283
Asn Glu c-1" Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr AAT GAG GAA TGT T-E TTC CTG GAA AGG TTG GAG GAA MC CAT TX
Asn Thr ~yr AX ACC TAC
317
Ile Ser Lys Lys His Ala Glu Lys His Trp Phe "al Gly Leu Lys Lys As" sly ATX TCC A&G MG CAT GCA GAG AAG CAT TGG TTC GTT GGT CTC AAG AAG .&AC GGA
391
Ar¶ Ser LYS Leu Gly Pro Arg Thr His Phe Gly Gln Lys Ala Ile Leu Phe LOU AGG TCT AM CTC GGT CCT CGG ACT CAC TTC GGC CAG AAA GCC ATc TTG TIT CTC
445
Pro Leu Pro v.31 Sex ser
Asp
RGGMGiCC~GGATGATGTCXGtTGGTGGAACTGGGG
1230
CMAGTPCATTTGGGTCTGGGGAGTGGGAGTGATA -
1102
~CAGTGGGTCTGTGGTGGAG~CCCAITTCTTACAACC
1518
kCTCCACCCCC~CACATTI\AATAGCXAGGTCCCTAXZACCC~GA CCCCATGCCCCATGGATCTTCCTCCAC'PCAGGCAGTPA
1590 1662
Fig.4.Nucleotide and amino acid seauences of bovine aFGF penomic clone FR2.1.The clone was sequenced by the chain termination method with double stranded DNmen%%ed by alkaline procedures. The first nucleotide corresponds to the ATG (Halley et al. 1989). The consensus polyadenylation signal sequences AATAAA are boxed. The poly A stretch is indicated with a wavy line. The 3’ destabilization motifs ATTT and TATT are underlined. 948
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The sequence of the 3’ UTR showed 36 ATTT and 23 TATT motifs and several classicaland unusual polyadenylation sites (23). The two classicalones are located at positions 2837 and 2986; the last one followed by an internal genomic poiy A strech. To determine which of these sites are used by the 471 and M4 clones, an RNAse protection assaywas performed using different fragments of the 3’ UTR (FigS). RNA probes containing the 2 classical polyadenylation sites were produced by transcription of the clone 471 and hybridized with poly A+ RNA from bovine brain and retina. One major protected fragment of 420 bp corresponding to the AATAAA at position 2986 was protected by both RNAs. Thus the M4 clone corresponds to the major aFGF transcript. A faint band corresponding to the full length probe was also visible. Additional RNAse protection assays showed that the RNA probe SspI-Pstl was also completely protected suggesting that the clone 471 corresponds to a minor aFGF transcript ending further downstream from the PstI site. 949
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2345
-
&
*
3’ END OF FR2.1
GENOMIC
?
a
e
420 340
CLONE)
-
RNA antisens probe protected fragment
Fig.5. RNAseurotectionassav The cDNA clone 471 (2577-3492) was subcloned in bluescriut KS(-). After linearization at the 5’ end, the antisense probe corresponding to this clone was produced by T7 RNA polymerase (BRL) in presence of (32P)UTP (Amersham). The restriction fragment KpnI-Pstl from genomic clone FR2.1 was subcloned in bluescript KS(-). After linearization by SspI (4098), antisense RNA corresponding to the PstI-SspI (4098-4378) fragment was produced. 2 ug of polyA+ mRNA isolated from bovine retina (lanes 2,3) and brain (lanes 4,s) were hybridized at 48°C with IO7 cpm antisense RNA 471 (3,5) and antisense RNA SspI-Pstl (2,4). Digestion with Tl and A RNAse was performed using an RNAse protection assay kit (ORION). The sizes of the protected fragments were determined using double stranded DNA sequencing of KnpI-PstI genomic subclones with specific synthetic oligonucleotides (lane 1, GATC). The arrows indicate the sizes of Mae111 and Sau3A bluescript restriction fragments used as markers.
DISCUSSION In the bovine the most highly expressed aFGF transcript is 4.2 kb, minor mRNAs of 1 kb and 9.9 kb are also detected in bovine brain and retina. In that species a 2.5 kb mRNA encoded by the 3’ end of the untranslated region was also expressed in these two tissues (13). The characterisation of the genomic and several cDNA clones shows that the 2.5 kb message is not encoded by the aFGF gene. We do not know the identity of this transcript that is expressed both in the brain and retina.The open reading frame which hybridized to it, is a result of a cloning artefact occurring inside the ORF 2 during the construction of the library. We observed such events in commercial and laboratory prepared libraries during the isolation of different genes. This fact implies that, specially for transcripts which have unusual long untranslated regions. several cDNAs from different speciesshould be analyzed and compared. In bovine, the coding region of bovine aFGF is interrupted by two large introns leading to a 101 bp middle exon. The exon 2 has the same size and the same 3’ and 5’ inuon-exon junctions as the other members of the aFGF family. The 3’ UTR of the 4.2 kb m&VA is 2.5 kb impliing a 1.2 kb 5’
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UTR. Such long UTR are unusual and probably involved in control of transcription of the aFGF gene. The classical poly adenylation site AATAAA used in the 4.2 kb mRNA is immediately followed by a polyA stretch which is not found in the human gene. Downstream from this polyA site, the terminator consensus sequencesnecessary for the cleavage of the transcripts (23) are not present, while such sequences are used in human to generate three minor transcripts at minor consensus polyadenylation sites (13). A minor transcript, corresponding to the clone 471, has a 3’UTR of at least 3.9 kb since the corresponding polyadenylation signal has not been identified in the genomic clone. This 3’UTR is the longest one actually reported for aFGF. The steady state levels of aFGF is very low which may result from a low level of transcription and/or from instability of the message. In addition to the multiple polyadenylation sites the 3’UTR also contains potential destabilization elements, As in human the motif ATITA is repeated 6 times within 780 bp of the 3’UTR. Three of them are located at the same positions in human and bovine species. 32 ATTT motifs are also present in the bovine 3’UTR; this sequence is repeated 24 times in human and only 2 times in the rat. These sequenceshave been shown to destabilize lymphokines, cytokines and proto-oncogene mRNAs (24) and may be involved in controlling the steady state levels of aFGF mRNA (25). On the other hand the role of 3’ UTR in controlling the transcription of one member of the FGF family (hst) has been established. Such mechanisms could also be used for the aFGF gene. Experiments are currently in progress to test this hypothesis.
ACKNOmEDGMENTS
We are grateful to He& Coet for photographic assistance, to Alain HaslC for drawings and David Hicks for hepful discussions. This work was supported by fellowships from Ministere de la Rechercheet de la Technologie and from the Fondation IFSEN.
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