Analysis of Turnover and Translation Regulatory RNA-Binding Protein Expression through Binding to Cognate mRNAs

Abstract

ABSTRACT RNA-binding proteins (RBPs) that associate with specific mRNA sequences and function as mRNA t urnover and t ranslation r egulatory (TTR) RBPs are emerging as pivotal posttranscriptional regulators of gene expression. However, little is known about the mechanisms that govern the expression of TTR-RBPs. Here, we employed human cervical carcinoma HeLa cells to test the hypothesis that TTR-RBP expression is influenced posttranscriptionally by TTR-RBPs themselves. Systematic testing of the TTR-RBPs AUF1, HuR, KSRP, NF90, TIA-1, and TIAR led to three key discoveries. First, each TTR-RBP was found to associate with its cognate mRNA and with several other TTR-RBP-encoding mRNAs, as determined by testing both endogenous and biotinylated transcripts. Second, silencing of individual TTR-RBPs influenced the expression of other TTR-RBPs at the mRNA and/or protein level. Third, further analysis of two specific ribonucleoprotein (RNP) complexes revealed that TIA-1 expression was controlled via HuR-enhanced mRNA stabilization and TIAR-repressed translation. Together, our findings underscore the notion that TTR-RBP expression is controlled, at least in part, at the posttranscriptional level through a complex circuitry of self- and cross-regulatory RNP interactions.