Alternative Splicing of the Human Estrogen Receptor Alpha Primary Transcript: Mechanisms of Exon Skipping

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.