The role of the 3' untranslated region in the regulation of tyrosine hydroxylase transcript stability

The expression and activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is controlled at multiple levels. In addition to transcriptional regulation, recent work also points to control of tyrosine hydroxylase message stability as a potentially important mechanism for modulating synthesis of tyrosine hydroxylase mRNA. In particular, the binding of cytosolic proteins to the tyrosine hydroxylase 3' untranslated region has been correlated with enhanced tyrosine hydroxylase transcript stability in PC-12 cells during hypoxia, and may influence message turnover under normoxic conditions as well. A deletional mutagenesis approach was applied in the present studies to directly test the role of the tyrosine hydroxylase 3' untranslated region in the regulation of tyrosine hydroxylase mRNA stability under basal, normoxic conditions. Chimeric tyrosine hydroxylase constructs were prepared in which the coding region from the rat tyrosine hydroxylase gene was recombinantly linked to rat wild type or mutated 3' untranslated regions. In one mutation, essentially all of the 3' untranslated region was deleted, while in the other, the deleted sequence was replaced with an identical length of bacterial sequence from the lacZ gene. Each construct was stably expressed in the human BE(2)-C catecholaminergic cell line under the transcriptional control of a tetracycline-repressible transactivator. Treatment of cells with the tetracycline analog doxycycline inhibited the synthesis of the chimeric mRNAs and the ensuing decline in message levels provided an index of transcript stability. Chimeric tyrosine hydroxylase mRNA levels were quantified using either RNase protection or competitive reverse transcriptase-polymerase chain reaction assays. Results showed a small increase in the stability of transcripts with a deleted untranslated region compared to transcripts containing the wild type untranslated region. In contrast, the transcripts containing the lacZ sequence were destabilized with respect to the wild type transcript. These data are consistent with a role for the tyrosine hydroxylase 3' untranslated region in the metabolism of tyrosine hydroxylase mRNA. Use of the tetracycline-repressible transactivator system also produced half-lives for the chimeric tyrosine hydroxylase message that were considerably faster than previously reported for rat tyrosine hydroxylase mRNA. Comparisons of chimeric tyrosine hydroxylase transcript decay following doxycycline or actinomycin D exposure in parallel cultures of the same clonal cell line indicated that consistently faster rates of decay were determined using the tetracycline-repressible transactivator approach. This difference was not caused by the design of the chimeric constructs, nor did it result from the use of doxycycline to suppress chimeric tyrosine hydroxylase transcription. These data suggest that the half-life of the tyrosine hydroxylase transcript may be significantly shorter than previously estimated.