Identification of C-terminally and N-terminally truncated estrogen receptor α variants in the mouse.
We re-examined mouse ERα mRNA variants using rapid amplification of cDNA ends (RACE) and RT-PCR. Our analysis showed the presence of several mRNA variants containing unique 5'- or 3'-nucleotide sequences. We mapped the cDNA sequences on the mouse genome, and identified four novel 3'-terminal and 5'-leader exons in the intronic region between exons 4 and 5. RT-PCR analysis revealed that the expression patterns of the C-terminally truncated ERα products (CTERPs) were similar to that of Wild-type ERα and that the N-terminally truncated ERα products (NTERPs) appeared to have different expression profiles. Moreover, we constructed expression vectors and analyzed the subcellular localization and the transcriptional activation abilities of the variant proteins in transfected HEK293 cells using immunocytochemistry and luciferase reporter assay. The CTERP variants localized in the nuclei and constitutively activated estrogen response element (ERE)-driven promoters, while the NTERP variant was located in the extra-nuclear regions and had no ability to activate the ERE promoters in the presence or absence of 10 nM estradiol. Our results indicate that the mouse ERα gene is more complex than previously thought in terms of genomic organization and that alternative splicing and alternative usage of intronic promoters contribute to the remarkable diversity of ERα mRNAs and proteins.
Department of Physiology, Nippon Medical School, Tokyo, Japan. hirotaka@nms.ac.jp
Hirotaka
H
Ishii
Yuri
Y
Shoda
Kentaro
K
Yomogida
Tomohiro
T
Hamada
Yasuo
Y
Sakuma
D017398:Alternative Splicing; D000818:Animals; D002874:Chromosome Mapping / Q000379:methods; D047628:Estrogen Receptor alpha / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019105:Random Amplified Polymorphic DNA Technique; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D047628:Estrogen Receptor alpha; D020033:Protein Isoforms; D012333:RNA, Messenger