REGULATED SPECIFIC PROTEIN-BINDING TO A CONSERVED REGION OF THE 3'-UNTRANSLATED REGION OF THYROTROPIN BETA-SUBUNIT MESSENGER-RNA

Thyroid hormone (T-3) regulates the expression of rat TSH beta-subunit (TSH beta) mRNA, in part, at the posttranscriptional level, by reduci ng the half-life of TSH beta mRNA, The mechanism(s) mediating this alt eration in mRNA stability are unknown, but previous work indicates tha t labile protein(s) are involved. The majority of cis-acting elements identified to date that have been implicated in the regulated destabil ization of mRNAs have been located in the 3'-untranslated region (3'-U TR) of the mRNA. The 3'-UTR of rat, murine, and human TSH beta mRNA is highly conserved, and within this region is a 12-nucleotide consensus sequence, which is shared by the 3'-UTR of several other genes with u nstable mRNAs. We reasoned that this homologous region could represent a binding motif for specific trans-acting RNA-binding protein(s), and that identification and characterization of such trans-acting factor( s) may provide critical insight into the mechanisms underlying T-3-ind uced changes in TSH beta mRNA stability. Utilizing the RNA electrophor etic mobility shift assay and analysis of UV cross-linked RNA-protein complexes, a cytoplasmic trans-acting factor of approximately 80-85 ki lodaltons was identified from rat pituitaries and several cell lines t hat binds in a sequence-specific manner to the 3'-UTR of rat TSH beta mRNA. Using competitive antisense oligonucleotides, the predominant bi nding site was mapped to the first 41 nucleotides of the 3'-UTR, which includes the consensus region. However, sequence upstream of the cons ensus was also shown to be important for binding. Using RNA electropho retic mobility shift assay, two mRNAs containing sequence homology wit h the consensus region, c-erbA alpha-2 and a rat ferritin pseudogene, were shown to specifically compete with rat TSH beta mRNA for binding of this factor. Remarkably, the binding activity of this factor was re gulated positively by T-3 within 4 h, but only with rat pituitary extr acts. These data suggest that in addition to binding rat TSH beta mRNA in a sequence-specific and T-3-regulated manner, this novel trans-act ing RNA-binding protein may also bind to other cytoplasmic mRNAs invol ved in diverse intracellular processes.