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The isolation and characterization of several new human estrogen receptor-alpha (hERalpha) mRNAs are described. Together with those previously identified, they give rise to a total of six hERalpha mRNA isoforms (A-F hERalpha mRNAs). Produced from a single hERalpha gene by multiple promoter usage, all these transcripts encode a common protein but differ in their 5'-untranslated region as a consequence of alternative splicing of five upstream exons (1B-1F). RT-PCR and S1 nuclease mapping analysis of these different hERalpha mRNA isoforms revealed a differential pattern of expression of the hERalpha gene in human tissues and cell types. The A hERalpha mRNA is the main isoform detected in mammary glands or in the tumor cell lines derived from this tissue. In endometrium, the predominant forms are the A and C hERalpha mRNA isoforms, whereas the C and F hERalpha mRNA isoforms are the major forms detected in ovary. Finally, high levels of the E hERalpha mRNA isoform are restricted to the liver with an increased expression in females. Taken together, our results demonstrate that the hERalpha gene is a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner.

MOTIVATION: The untranslated regions (UTRs) of mRNA upstream (5'UTR) and downstream (3'UTR) of the open reading frame, as well as the mRNA precursor, carry important regulatory sequences. To reveal unidentified regulatory signals, we combine information from experiments with computational approaches. Depending on available knowledge, three different strategies are employed. RESULTS: Searching with a consensus template, new RNAs with regulatory RNA elements can be identified in genomic screens. By this approach, we identify new candidate regulatory motifs resembling iron-responsive elements in the 5'UTRs of HemA, FepB and FrdB mRNA from Escherichia coli. If an RNA element is not yet defined, it may be analyzed by combining results from SELEX (selective enrichment of ligands by exponential amplification) and a search of databases from RNA or genomic sequences. A cleavage stimulating factor (CstF) binding element 3 of the polyadenylation site in the mRNA precursor serves as a test example. Alternatively, the regulatory RNA element may be found by studying different RNA foldings and their correlation with simple experimental tests. We delineate a novel instability element in the 3'UTR of the estrogen receptor mRNA in this way. AVAILABILITY: Strategy, methods and programs are available on request from T.Dandekar. CONTACT: dandekar@embl-heidelberg.de

Primer extension is one of the most common methods used to measure the amount and size of RNAs. We demonstrate that the sensitivity and the specificity of this method are improved considerably by using a highly-labeled single-stranded DNA generated from a biotinylated single-stranded DNA template, as a long specific primer in the reverse transcription reaction. This new approach allows the detection of transcripts with a low expression level from microgram quantities of total RNA.