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

Graceffo et al., International Journal of Molecular Sciences, 2024 set out to solve a measurement problem. The thyroid hormone receptor alpha gene, THRA, is spliced into two isoforms, THRA1 and THRA2, whose balance decides how a cell answers triiodothyronine, and the two proteins are so alike that no antibody separates them. The authors built a plasmid reporter, pCMV-THRA-RFP-EGFP, carrying the human THRA region from exon 7 to the 3 prime untranslated region of exon 10, with red fluorescent protein in place of the exon 9b stop codon and green fluorescent protein in place of the exon 10 stop codon. A cell that splices to THRA1 turns red, a cell that splices to THRA2 turns green, and single cells can be followed live.

A reporter is only as good as its response to a known perturbation, and that is where the AUM product comes in. An AUMblock sdASO aimed at the exon 10 splice acceptor of THRA was added free to the medium of COS1 cells at 1 μM, 24 hours after seeding and 24 hours before the reporter plasmid went in. No transfection reagent was used for the oligonucleotide. A fluorescently tagged scrambled control showed it entered 98.4% of the cells on its own.

The result was complete. In untreated cells the two isoform signals sat at 52% red and 48% green; in treated cells the green signal was gone and the split read 100% red and 0% green. A scrambled control oligonucleotide changed nothing. RT-qPCR confirmed that neither the reporter nor the treatment altered how much THRA the cells transcribed, so what moved was which isoform was made. The authors then used the reporter for hour by hour time-lapse imaging of single cells, tracking isoform expression across 48 hours in COS1 cells and 16 hours in N2A neuroblastoma cells, including across one cell division.