A new isoform of the human estrogen receptor-alpha (hER-a) has been identi®ed and characterized. This 46 kDa isoform (hERa46) lacks the N-terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERa66). hERa46 is encoded by a new class of hER-a transcript that lacks the ®rst coding exon (exon 1A) of the ER-a gene. We demonstrated that these D1A hER-a transcripts originate from the E and F hER-a promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERa46 showed that, in a cell context sensitive to the transactivation function AF-2, this receptor is an effective ligand-inducible transcription factor. In contrast, hERa46 is a powerful inhibitor of hERa66 in a cell context where the transactivating function of AF-1 predominates over AF-2. The mechanisms by which the AF-1 dominantnegative action is exerted may involve heterodimerization of the two receptor isoforms and/or direct competition for the ER-a DNA-binding site. hERa66/ hERa46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERa46 in cellular proliferation.

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Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

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.