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Recently, we have isolated the untranslated first exon 'exon ON' of the rat estrogen receptor (ER) gene from the liver by the use of the 5'-rapid amplification of cDNA ends (5'-RACE) method. To investigate the existence of other untranslated first exon(s), we further analyzed the 5'-untranslated region (UTR) of ER mRNA in the rat liver in this study. Total RNA from the livers of 8-week-old male Wistar rats was subjected to 5'-RACE with the antisense primers located in exon 1 of the rat ER gene. The inserts of four clones (clones 3, 4, 7 and 8) were sequenced. The nucleotide sequences of the clones revealed the existence of a previously unidentified untranslated first exon (we termed it 'exon 0S') which was spliced onto exon 1 of the rat ER mRNA. The distribution of ER mRNA containing 'exon 0S' (ER mRNA (0S-I)) in several brain regions and various peripheral tissues of 8-week-old male and female Wistar rats was further analyzed by the use of the reverse transcription-polymerase chain reaction. ER mRNA (0S-l) was found to be widely distributed in the rat brain and peripheral tissues. The distribution of the message was different from that of ER mRNA containing exon 0 (the first reported 5'-UTR form of rat ER mRNA) or of ER mRNA with exon ON which was reported in our recent report. These results indicate that (1) 'exon 0S' is a novel untranslated first exon of the rat ER gene, (2) rat ER mRNAs possess at least three forms of 5'-UTRs which are exon 0, exon ON, and exon 0S, (3) the tissue specific expression of ER is regulated, at least in part, by the usage of differential promoters in the rat.

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Abstract. Presence of alternatively spliced-estrogen receptor (ER) mRNA variants has been revealed in the breast cancer tissues. The ER variants transcribed from these mRNA variants were supposed to cause changes in the estrogen responsiveness of breast cancer. Although uterine endometrial cancer also has an estrogen-dependent profile, these ER mRNA variants have not yet been reported in the tumor. In the present study, we attempted to detect the exon 7 deletion-(del.7-) and exon 5 deletion (del.5) ER mRNA variants in normal human uterine endometrium (hEM) and uterine endometrial cancer tissue (hEC) by the use of reverse transcription-polymerase chain reaction-Southern blotting (RT-PCR-SB) with the PCR primers: hE4 (forward), hE6 (reverse), and hE8 (reverse), which were located in exons 4, 6, and 8, respectively. Two major products were generated from RNAs of both hEM and hEC with primers hE4 and hE8. The nucleotide sequence of the longer product was identical to exon 4-8 of human ER cDNA, whereas that of the shorter one completely deleted exon 7. Moreover, when the RT-PCR was done with the primers hE4 and hE6, the shorter product lacking exon 5 was detected with the longer one having the same sequence as exon 4-6 of human ER cDNA. Since the RT-PCR-SB with primers hE4 and hE8 produced a very low or undetectable level of the signals corresponding to del.5 ER mRNA variant, the level of del.7 ER mRNA variant seemed to be higher than that of del.5 ER mRNA variant. These results strongly suggested that both de1.7-and del.5 ER mRNA variants exist in the normal uterine endometrium as well as in endometrial cancer. The ER variants, possibly expressed in these tissues, may play a physiological and/or pathological role.