Endocrine Abstracts

In humans only thyroid hormone (T3) receptor (TR) beta1 and beta2 mRNAs have been identified, whereas alternative splicing generates recently characterised beta3 and deltabeta3 transcripts in rat and further N-terminal isoforms in other species. We investigated whether additional transcripts arise from the human TRbeta gene by 5'-RACE. Five distinct TRbeta1 transcripts were isolated, including the previously described TRbeta1 mRNA and four novel 5'-untranslated region (5'-UTR) variants, but TRbeta3 and deltabeta3 were not identified. We isolated genomic clones and determined that the TRbeta1 5'-UTR contains nine exons, including four that are novel. Nevertheless, the structure of the translated TRbeta1 protein remains unaffected by the alternative 5'-UTRs. The variant mRNAs were expressed in temporo-spatial specific patterns as revealed by Northern analysis using exon-specific probes. To investigate their effects on gene expression, 5'-UTRs were cloned upstream of luciferase into pBluescript and pGL3 vectors. Transcription of pBluescript constructs and translation of equal amounts of RNA revealed that each 5'-UTR reduced luciferase activity (to between 1.8plus/minus0.5% and 20.6plus/minus7.3% of control), indicating that they differentially regulate protein translation efficiency. In transfections using pGL3 constructs, each 5'-UTR reduced luciferase activity to between 34plus/minus6% and 74plus/minus9% (COS-7 cells) or 17plus/minus1% and 89plus/minus9% (JEG-3 cells) of control, indicating both cell- and 5'-UTR-specific effects on gene expression. These effects were not regulated by T3. To investigate the influence of each 5'-UTR on mRNA expression, RNase protection analysis was performed. Luciferase mRNA was either increased (between 2.3plus/minus0.6 and 2.9plus/minus0.6 fold) or unaffected relative to control and there was no correlation between the effect of each 5'-UTR on mRNA expression compared to its effect on luciferase activity (r=0.262), indicating independent actions on mRNA expression and protein translation. These data indicate that temporo-spatial expression of human TRbeta1 5'-UTR variants may regulate T3-responsiveness of individual tissues by controlling receptor protein concentration.