Bichromatic Splicing Detector Allows Quantification of THRA1 and THRA2 Splicing Isoforms in Single Cells by Fluorescent Live-Cell Imaging

Abstract

Thyroid hormone receptor alpha (THR alpha) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development, metabolism, and reproduction. The coding gene, THRA, has two major splicing isoforms in mammals, THRA1 and THRA2, which encode THR alpha 1 and THR alpha 1, respectively. The better characterized isoform, THR alpha 1, is a transcriptional stimulator of genes involved in cell metabolism and growth. The less well-characterized isoform, THR alpha 2, lacks the ligand-binding domain (LBD) and may act as an inhibitor of THR alpha 1 activity. Thus, the ratio of THR alpha 1 to THR alpha 2 isoforms is critical for transcriptional regulation in various tissues and during development and may be abnormal in a number of thyroid hormone resistance syndromes. However, the complete characterization of the THR alpha isoform expression pattern in healthy human tissues, and especially the study of changes in the ratio of THR alpha 1 to THR alpha 2 in cultured patient cells, has been hampered by the lack of suitable tools to detect the isoform-specific expression patterns. Therefore, we developed a plasmid pCMV-THRA-RFP-EGFP splicing detector that allows the visualization and quantification of the differential expression of THRA1 and THRA2 splicing isoforms in living single cells during time-lapse and perturbation experiments. This tool enables experiments to further characterize the role of THR alpha 2 and to perform high-throughput drug screening. Molecules that modify THRA splicing may be developed into drugs for the treatment of thyroid hormone resistance syndromes.