Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame

In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified . This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobilit y shift assay to specifically interact with lutropin receptor RNA sequences . The present studies have examined the specificity of lutropin receptor mR NA recognition by LRBP-1 and mapped the contact site by RNA footprinting an d by site-directed mutagenesis. LRBP-1 was partially purified by cation-exc hange chromatography, and the mRNA binding properties of the partially puri fied LRBP-1 were examined by RNA gel electrophoretic mobility shift assay a nd hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 bin ding site is located between nucleotides 203 and 220 of the receptor open r eading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC -X-7-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding c ompetitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutage nesis of the cytidine residues contained within the LRBP-1 binding site dem onstrated that all the cytidines in the bipartite sequence contribute to LR BP-1 binding specificity. Additionally, RNA gel electrophoretic mobility su pershift analysis showed that LRBP-1 was not recognized by antibodies again st two well-characterized poly(rC) RNA binding proteins, alpha CP-1 and alp ha CP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRB P-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lu tropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.