The multiple untranslated first exons system of the human estrogen receptor beta (ERβ) gene

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Abstract

In order to investigate the regulatory mechanism of the expression of the human estrogen receptor β (ERβ) gene, we have analyzed the structure of the 5′-untranslated region of the ERβ mRNA in the normal uterine endometrium and liver using the 5′-rapid amplification of the cDNA ends method. The sequence analysis revealed the presence of the two isoforms of the ERβ mRNA containing the distinct 5′-untranslated regions. The genomic analysis revealed that the two isoforms of the message originated from the two distinct untranslated first exons, termed the exon 0K and exon 0N, which were spliced to the exon 1. We termed the two isoforms of the message the ERβ mRNA (0K–1) and ERβ mRNA (0N–1). We further analyzed the distribution of the ERβ mRNA (0K–1) and ERβ mRNA (0N–1) in the ejaculated spermatozoa, liver, uterine endometrium and myometrium, and peripheral leukocytes using the reverse transcription-polymerase chain reaction. The distributions of the two mRNA isoforms were different from each other. From these results, it is indicated for the first time that the expression of the human estrogen receptor β (ERβ) gene is regulated, at least in part, by the multiple untranslated first exons and promoters system.

Introduction

The tissue-specific expression of the human estrogen receptor α (ERα) gene has been reported to be regulated, at least in part, by the multiple untranslated first exons and promoters system in humans and rats. In humans, five untranslated first exons exist: the exon 0 [1], [2], [3], [4], exon Ca (the 5′-part of the ‘exon C’ reported by Grandien [5]), exon Ha [6], exon 10 [7], and exon E [6], and one untranslated second exon, exon Hb [6]. Moreover, six isoforms of the ERα mRNA having distinct 5′-untranslated regions are generated from these exons in a tissue-specific fashion: the messages with the exons 0–1, exons Ca–Hb–1 (the exons Ca–Hb corresponded to the ‘exon C’ (Grandien)), exons Ha–Hb–1, exons 10–1 exons E–1, and the transcript started from exon 1 [6], [7], [8], [9], [10]. Moreover, three untranslated first exons have been reported in rats: the exon 0 [11], exon 0S [12], which is the homologue of the human exon Ha, and exon 0N [13]. In addition, three isoforms of the ERα mRNA with the distinct 5′-untranslated regions are tissue-specifically generated from the exons; the messages with the exons 0–1, exons 0S–1 and exons 0N–1 [12], [13]. In spite of the above progress in the study of the gene expression of the ERα gene, few reports on the regulatory mechanism of the gene expression of the estrogen receptor β (ERβ) [11], [12], [13], [14], [15], [16], [17], [18], [19] are available so far. In the present communication, we have analyzed the structure of the 5′-untranslated region of the human ERβ mRNA in the ejaculated spermatozoa, liver, uterine endometrium and myometrium, and peripheral leukocytes.

Section snippets

Tissues and RNA extraction

The ejaculated spermatozoa (Sp) and peripheral leukocytes (WBC) were obtained from a healthy volunteer. The sperm sample was prepared by the swim-up method and no contamination with non-sperm cells such as leukocytes was confirmed by microscopic examination. The uterine endometrium in the proliferative phase (Em) and myometrium (Mm) were collected from a resected uterus during an operation for uterine leiomyoma after obtaining informed consent. The liver tissue (Li) was sampled from the

Results

Using the 5′-RACE method, six and ten positive clones were obtained from the uterine endometrium (Em) and liver (Li). The six Em clones contained the various lengths of the 5′-untranslated region (UTR), which were parts of the human ERβ cDNA reported by Moore et al. [19], termed the ‘ERβ cDNA (Moore)’. The nucleotide sequence of the longest clone R258 and 5′-ends of the other clones are indicated in Fig. 1B. On the other hand, the sequence of the 5′-side of the 5′-UTR of the two clones from the

Discussion

The 5′-RACE analysis of the RNA of the uterine endometrium and liver and the analysis of the genomic DNA demonstrated that the two distinct untranslated first exons, termed the exon 0K (Fig. 2A) and exon 0N (Fig. 2B), existed in the upstream region of the exon 1 (Fig. 2C) of the human ERβ gene, and that two isoforms of the transcripts containing two distinct 5′-UTR, termed the ERβ mRNA (0K–1) and ERβ mRNA (0N–1), were generated from these two untranslated exons. The order of the two

Acknowledgements

The authors gratefully acknowledge the technical assistance of Ms Michiko Yoneyama. This work was supported by grants No. 08671879 and No. 11671605 to SH from Japanese Ministry of Education.

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