The Novel Untranslated First Exon “Exon 0N” of the Rat Estrogen Receptor Gene

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 849–854 (1996)ARTICLE NO. 1262

The Novel Untranslated First Exon ‘‘Exon 0N’’of the Rat Estrogen Receptor Gene

Shuji Hirata,*,1 Tomoko Koh,* Naoko Yamada-Mouri,* and Junzo Kato*,†

*Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma,Yamanashi 409-38, Japan; and †Section on Genes and Bioregulation, Department of Culture and Information,

Faculty of Informatics, Teikyo Heisei University, Ichihara, Chiba, Japan

Received July 16, 1996

The 5*-untranslated region (UTR) of the estrogen receptor (ER) mRNA in the rat liver was analyzed bythe use of the 5*-rapid amplification of the cDNA ends (5*-RACE) method. The nucleotide sequence ofone of the positive RACE clones (clone 9) revealed that the existence of the novel untranslated first exon(termed ‘‘exon 0N’’) being spliced onto the exon 1 of the rat ER mRNA. We further analyzed the distributionof the ER mRNA containing the ‘‘exon 0N’’ (ER mRNA (0N-1)) and the ER mRNA containing thepreviously reported exon 0 (ER mRNA (0-1)) in the rat brain and peripheral tissues. In contrast to the widedistribution of the ER mRNA (0-1), the distribution of the ER mRNA (0N-1) was almost limited in theperipheral tissues. These results indicate that the ‘‘exon 0N’’ is the novel untranslated first exon of the ratER gene, and the tissue specific expression of the ER is regulated, at least in part, by differential promoterusage in the rat. q 1996 Academic Press, Inc.

Recent reports on the human estrogen receptor (hER) gene have revealed that the ER mRNAis transcribed from at least three promoters, the proximal promoter P-1, the distal promoterP-0 and the far distal promoter P-C (1-4). The P-1, P-0 and P-C locate upstream the exon 1,the ‘‘exon 0’’ which is also named ‘‘the exon 1*’’ by Keaveney et al. (1) of the hER gene,and the recently identified untranslated exon which is termed ‘‘the exon C’’ by Grandien (5),respectively. Since the exon 0 and the exon C are the untranslated exon being spliced ontothe 5*-untranslated region (5*-UTR) of the exon 1, the ER mRNA transcribed from P-0 andP-C encode the same ER protein as the message transcribed from P-1. The studies on theactivity of the promoters P-1 and P-0 in the human breast cancer cells, normal breast tissue,uterine tissue, and osteoblast cells indicate that the tissue specific expression of the ER isregulated, at least in part, by the differential promoters usage (6-8).

In the rat, only one form of the 5*-UTR of the ER cDNA which was cloned from the uterushas been reported (9). Analysis of the sequence homology between the rat and human cDNAsshowed that the rat ER cDNA contained the exon 0 and therefore the clone originated fromthe ER mRNA transcribed from the distal promoter P-0 (1). Since the other 5*-UTRs of therat ER cDNA has been not reported so far, it has been unclear whether the differential usageof the multiple promoters involves in the regulation of the tissue specific gene expression ofthe ER in the rat. To solve this issue, we analyzed the 5*-UTR of the ER mRNA in the ratliver by the use of the 5*-rapid amplification of the cDNA ends (5*-RACE) method (10) withthe antisense primers located in the exon 1 of the rat ER gene.

1 Address all correspondence and requests for reprints to Dr. Shuji Hirata, Department of Obstetrics and Gynecology,Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, 409-38, Japan. Fax: 011-81-552-73-6746; e-mail: [email protected].

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

849

AID BBRC 5277 / 6907$$1101 08-06-96 09:00:34 bbrcas AP: BBRC

Vol. 225, No. 3, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Location and sequences of the PCR and RACE primers. The sequence of the primers rE0s (forward),rE1as (reverse) and rE1RACEas (reverse) were derived from the rat ER cDNA sequence (8). The sequence of theprimer rE0Ns (forward) was derived from the nucleotide sequence of the RACE clone 9. The sequences of the primersRACE1 and RACE II were identical to those reported by Frohman et al. (9). In order to facilitate the subcloning, afew substitutions of nucleotide were introduced in the primers rE1as, rE1RACEas, rE0s and rE0Ns0s at the positionsindicated by the underlines. The dashed line indicates a splicing acceptor site on the exon 1.

MATERIALS AND METHODSTissues. Eight-week-old Wistar strain male and female rats were used. Several regions of the brain which were

anterior hypophysis (AP), hypothalamus and preoptic area (HPOA), amygdala (AMY), cerebral cortex (CC) andcerebellum (Ce) were dissected from three male rats as described previously (11), and various peripheral tissues whichwere liver (Li), kidney (Ki), spleen (Sp), adrenal gland (Ad), small intestine (Is), large intestine (Il), heart (He),adipose tissue (Fa), and testis (Te) from three male rats, and uterus (Ut) and ovary (Ov) from three female rats werealso dissected.

RNA extraction. Total RNA was extracted from tissues according to the procedure of Chirgwin et al. (12) withminor modifications; in brief, tissues were homogenized in 4M guanidine isothiocyanate solution and the total RNAwas pelleted through a 5.7M cesium chloride cushion by ultracentrifugation at 35,000 rpm (Beckman, SW50.1 rotor)for 12h at 207C. The RNA concentration was determined by OD260.

5*-rapid amplification of the cDNA ends (5*-RACE). The 5*-RACE was carried out essentially as described byFrohman et al. (10); briefly, 1mg of the total RNA of the liver was subjected to the reverse transcription (RT) withthe primer rE1as (Fig. 1) under the condition described bellow, followed by poly A tailing with 15 units of terminaldeoxynucleotidyltransferase (Bethesda Research Laboratories, Gaitherburg, MD, USA) at 377C for 15min. The polyA tailed cDNA was subjected to the polymerase chain reaction (PCR) with primers rE1RACEas, RACE1, and RACE2(Fig. 1). The reaction was carried out for 40 cycles at 947C for 1min, 557C for 1min and 727C for 1min. The amplifiedproduct, digested by Xba I and Eco RI, was subcloned into pBS M13/ vector (Stratagene, LaJolla, CA, USA). TheRACE library were screened by Southern blotting with the 32P labeled rat ER (exons 0-1) cDNA probe synthesizedas described bellow. We isolated nine positive clones, and the nucleotide sequence of the insert of the clone 9 wasdetermined by the dideoxy method by Sanger et al. (13) with a Sequenase DNA sequencing kit (USB, Cleveland,OH, USA).

PCR primers. The locations and sequences of the PCR primers are shown in Fig.1. The sequence of the primersrE0s (forward), rE1as (reverse) and rE1RACEas (reverse) were derived from the rat ER cDNA sequence (8). Thesequence of the primer rE0Ns (forward) was derived from the nucleotide sequence of the RACE clone 9. The expectedlengths of the RT-PCR products with primers rE0Ns/rE1as and rE0s/rE1as were 305bp and 271bp, respectively. Sincethese primers considered to flank the region including the intron on genomic DNA (Fig.1), the expected lengths ofthe RT-PCR products could be generated only from respective mRNAs, and not from genomic DNA.

Reverse transcription (RT). Total RNA from each tissue was reverse transcribed to synthesize the single strandedcDNA. 200ng of the total RNA from the tissues were incubated at 427C for 60min with 2 units of RAV-2 reverse

850



transcriptase (Takara, Kyoto, Japan) in a 10ml reaction volume containing 50mM Tris-HCl (pH8.3), 100mM KCl,10mM MgCl2 , 10mM dithiothreithol (DTT), 1mM each of dNTP and 10mM random hexadeoxynucleotide primer(Takara).

Polymerase chain reaction (PCR). A PCR was performed as recommended by the manufacturer (Perkin Elmer,Branchburg, NJ, USA) (14) with minor modification. Briefly, 1ml of the cDNA was amplified in a 10ml reactionvolume containing 0.25unit of Taq DNA polymerase (Perkin Elmer) and 1.5mM MgCl2 . The reaction wasperformed for 26 cycles (primers rE0s/rE1as) or 32 cycles (primers rE0Ns/rE1as) at 947C for 1min, 557C for1min and 727C for 1min.

Direct nucleotide sequencing. In order to confirm the specificity of the reaction, the RT-PCR products from uterineRNA with the primers rE0s/rE1as and the primers rE0Ns/rE1as were subjected to direct nucleotide sequencing. Thesequencing was carried out by the dideoxy method.

Southern blotting. Two microliter of the RT-PCR products from each tissue RNA were electrophoresed in 2.0%agarose gel and transferred onto a Nylon membrane (Hybond N/, Amersham, Buckinghamshire, UK) with 400mMNaOH as a transfer solution for 3h. The membrane was incubated in a prehybridization buffer containing 61 SSC(11 SSC: 15mM sodium chloride-1.5mM sodium citrate-pH7.0), 150mg/ml yeast total RNA, 1.0% sodiumdodecil-sulfate (SDS)) at 427C for 1h. Then the membrane was hybridized with the 32P labeled rat ER (exons 0-1) or rat ER(exons 0N-1) cDNA probes in the same buffer at 657C for 12h. The radioactive probes were synthesized by therandom priming method using the RT-PCR products from the uterus with the primers rE0s/rE1as or rE0Ns/rE1as asa template. The templates were confirmed to be the part of the rat ER cDNA by direct nucleotide sequencing. Thespecific activity of the probe was approximately 1.0 Ç 2.0 1 109 cpm/mgDNA. After hybridization, the membranewas washed under stringent condition. The hybridization signal was analyzed by a Bioimage Analyzing System,BAS2000 (Fuji Film, Tokyo, Japan).

RT-PCR blank. In order to examine whether contamination of reagents occurred in the present experiments, distilledwater was simultaneously subjected to RT-PCR (RT-PCR-blank). But no specific signal was obtained from the RT-PCR blank, indicating no contamination occurred in these experiments.

RESULTS AND DISCUSSION

The RACE library were constructed from the rat liver total RNA with the antisense primerslocated in the exon 1 of the rat ER cDNA. Nine positive clones were isolated by screeningof the clones with the rat ER (exons 0-1) cDNA probe and one of the clones (clone 9) wassubcloned and sequenced. The nucleotide sequence of the 291bp of the insert of the clone 9includes the 24bp of the primer RACE I and the 10bp of poly T in the 5* end, and the 23bpof the primer rE1RACEas in the 3* end (Fig. 2). The remaining sequence of the 234bp wascompared with the rat ER cDNA. The sequence includes the translation initiation site of therat ER gene and the A of the first codon ATG are assigned to nucleotide number /1. Thenucleotide sequence from nucleotide (nt) 071 to /60 corresponds to the part of the exon 1of the rat ER cDNA except one nucleotide substitution within the 5*-UTR. The G was foundat nt 028 in the present study instead of the A in the firstly reported rat ER cDNA (pRcER6)(9). We confirm that the nucleotide at this position is the G by the nucleotide sequencing ofthe rat genomic DNA (data not shown). However, the other sequence between nt 071 and/60 is identical to the pRcER6, indicating that the clone 9 originated from the transcript forthe rat ER. On the other hand, the region from nt 0184 to 072 of the clone 9 was differentfrom the upstream region from nt 072 of the pRcER6 which is suggested to be the exon 0of the rat ER gene by the analysis of the sequence homology with the human ER cDNA (1).Moreover, the region from nt 0174 to 072 did not show any homology with the correspondingpart of the firstly reported human ER cDNA (pOR8) which was indicated to be derived fromthe exon 1 of the human ER gene (1-3, 15, 16), and the exon C which was recently identifieduntranslated exon from the human liver (5). We further analyzed the upstream sequence of nt072 of the exon 1 of the rat ER gene by the genomic cloning, however, the sequence wasdifferent from that of the region from nt 0174 to 072 of the RACE clone 9 (data not shown).From these results, we conclude that the clone 9 originated from the ER mRNA containingthe novel untranslated first exon (we termed ‘‘exon 0N’’) which is spliced onto the 5*-UTRof exon 1 at nt 071.

851



FIG. 2. Nucleotide sequence of the RACE clone 9. The RACE library constructed from the rat liver total RNAwas screened by the rat ER cDNA (0–1) probe and one of the clones (clone 9) was sequenced. The nucleotidesequence of the 291bp includes the 24bp of the primer RACE I (indicated by the box) and the 10bp of poly T in the5* end, and the 23bp of the primer rE1RACEas in the 3* end (indicated by the box). The A of the first codon ATG(indicated by the double underline) are assigned to nucleotide number /1. The nucleotide sequence from nucleotide(nt) 071 to /60 corresponds to the part of the exon 1 of the rat ER cDNA except one nucleotide substitution withinthe 5*-UTR. The G (*)was found at nt 028 in the present study instead of the A in the reported rat ER cDNA (8).The region from nt 0174 to 072 (indicated by Italic) was different from the corresponding region of the reportedrat ER cDNA (1), and not similar to the corresponding part of the first reported human ER cDNA (pOR8) (14, 15).We conclude that the clone 9 originated from the ER mRNA containing the novel untranslated first exon (termed‘‘exon 0N’’) which is spliced onto the 5*-UTR of exon 1 at nt 071 (.). The location of the primer rE0Ns is underlined.

Furthermore, we investigated the distribution of the ER mRNA containing ‘‘exon 0N’’ (ERmRNA (0N-1)) and the ER mRNA containing exon 0 (ER mRNA (0-1)) in the several regionsof the rat brain and various peripheral tissues by the use of the RT-PCR with the primersrE0Ns/rE1as and rE0s/rE1as. The high levels of the ER mRNA (0N-1) were detected in someperipheral tissues which was the uterus, ovary, liver and kidney, with the low levels of themessage in the anterior hypophysis, spleen, large intestine, heart and testis. In the brain regionsexcept for the anterior pituitary, no signals of the ER mRNA (0N-1) could be detected (Fig.3A). On the other hand, the ER mRNA (0-1) was widely distributed in the rat brain regionsand peripheral tissues. The levels of the ER mRNA (0-1) were high in the anterior hypophysis,hypothalamus and preoptic area, amygdala, ovary and uterus with the lower but definite levelof the message in the other brain regions and peripheral tissues (Fig. 3B). The distribution ofthe ER mRNA (0-1) in the brain regions was similar with that of the message containingexons 4-6 which was reported previously by us (17), it was considered that the major transcriptof the ER might possess the exon 0 in the rat brain. The differential distribution of the ERmRNA (0N-1) and the ER mRNA (0-1) in the rat brain and peripheral tissues has indicatedthat the ER mRNA (0N-1) is transcribed from the unique promoter P-N which is specific forthe peripheral tissues and is not functional in the brain regions with the exception for theanterior hypophysis.

The presence of the ER mRNA transcribed from the proximal promoter P-1 has been notyet reported. By the study using the transgenic mouse, it has been considered whether onlydistal promoter P-0 is functional or the differences in the activity of P-0 and P-1 is very subtle

852



FIG. 3. The distribution of the ER mRNA (0N-1) and the ER mRNA (0-1) in the several regions of the rat brainand various peripheral tissues. 200ng of total RNA from various tissues from the 8-week-old male and female ratsindicated in Materials and Methods were subjected to the RT-PCR using primers rE0Ns/rE1as and rE0Ns/rE1as. ThePCR was performed for 26 cycles (primers rE0s/rE1as) or 32 cycles (primers rE0Ns/rE1as) at 947C for 1min, 557Cfor 1min and 727C for 1min. The signals of the RT-PCR products were analyzed by Southern blotting under thestringent condition. AP, anterior hypophysis; HP, hypothalamus and preoptic area; AMY, amygdala; CC, cerebralcortex; Ce cerebellum; Li, liver; Ki, kidney; Sp, spleen; Ad, adrenal gland; Is, small intestine; Il, large intestine; He,heart; Fa, adipose tissue; Te testis, were dissected from the male rats. Ut, uterus and Ov, ovary were dissected fromthe female rats, Bl, RT-PCR blank.

in the mouse (18). Thus, it had not been clarified whether the differential usage of the multiplepromoters regulated the ER gene expression in a tissue specific fashion in the rodents. Thisis the first report which revealed that the tissue specific expression of the ER in the rodentsis regulated, at least in part, by the differential usage of at least two promoters, P-0 and P-N.Future studies on the quantitive analysis of the ER mRNAs and the other untranslated firstexon (s) are essential to elucidate the precise mechanism of the multiple promoters system ofthe rat ER gene.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of Ms. Michiko Yoneyama. This work was supportedby grants No.01440069 to JK and No.08671879 to SH from Japanese Ministry of Education.

REFERENCES

1. Keaveney, M., Klug, J., Dawson, M. T., Nestor, P. V., Neilan, J. G., Forde, R. C., and Gannon, F. (1991) J. Mol.Endocrinol. 6, 111–115.

2. Keaveney, M., Klug, J., and Gannon, F. (1992) J. DNA. Seq. Map. 2, 347–358.3. Piva, R., Gambari, R., Zorzato, F., Kumar, L., and del Senno, L. (1992) Biochem. Biophys. Res. Commun. 183,

996–1002.4. Piva, R., Bianchi, G., Aguiari, G. L., Gambari, R., and del Senno, L. (1993) J. Steroid Biochem. Molec. Biol.

46, 531–538.5. Grandien, K. (1996) Mol. Cell. Endocrinol. 116, 207–212.6. Gradien, K. F. H., Berkenstam, A., Nilsson, S., and Gustafson, J.-A. (1993) J. Mol. Endocrinol. 10, 269–277.7. Gradien, K., Backdahl, M., Ljunggren, O., Gustafson, J.-A., and Berkenstam, A. (1995) Endocrinology 136,

2233–2229.8. Weigel, R. J., Crooks, D. L., Iglehart, D., and deConinck, E. C. (1995) Cell Growth Differentiation 6, 707–711.9. Koike, S., Sakai, M., and Muramatsu, M. (1987) Nucleic Acids Res. 15, 2499–2513.

10. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998–9002.

853



11. Kato, J., and Villee, C. A. (1966) Endocrinology 80, 567–575.12. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochem. 18, 5284–5299.13. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467.14. Kawasaki, E. S., and Wang, A. M. (1989) in PCR Technology (Ehrlich, H. A., Ed.), pp. 89–97, Stockton Press,

New York.15. Greene, G. L., Gilna, P., Waterfield, M., Baker, A., Hort, Y., and Shine, J. (1986) Science 231, 1150–1157.16. Green, S., Walter, P., Kumar, V., Bornert, J.-M., Argos, P., and Chambon, P. (1986) Nature 320, 134–139.17. Hirata, S., Osada, T., Hirai, M., Hagihara, K., and Kato, J. (1992) J. Steroid Biochem. Molec. Biol. 41, 583–587.18. Cicatiello, L., Cobellis, G., Addeo, R., Papa, M., Altucci, L., Sica, V., Bresciani, F., LeMeur, M., Kumar, V. L.,

Chambon, P., and Weisz, A. (1995) Mol. Endocrinol. 9, 1077–1090.

854


Documents

The Novel Untranslated First Exon “Exon 0N” of the Rat Estrogen Receptor Gene