RNA Recognition and Regulation of the AU-rich RNA Binding Proteins: HuR, TTP and BRF1

Posttranscriptional gene expression is controlled and coordinated by RNA binding proteins (RBPs), many of which recognize specific RNAs through cis-regulatory RNA elements. One of the most highly studied classes of cis-regulatory RNA elements is the AU-rich elements (AREs). AREs are bound by a class of RBPs called ARE binding proteins (ARE-BPs), of which there are over a dozen in humans including HuR, tristetraprolin (TTP) and butyrate response factors 1 and 2 (BRF1 and BRF2). TTP, BRF1 and BRF2 belong to a family of tandem C3H zinc finger proteins that destabilize ARE-containing mRNAs. HuR acts to enhance the stability and translation of ARE-containing mRNAs, a function that is rare among ARE-BPs. While each of these ARE-BPs regulates the expression of ARE-containing mRNAs, some ARE-BPs themselves are also encoded by ARE-containing mRNAs, raising the possibility that each of these ARE-BPs may regulate one another's expression. In order to determine how these ARE-BPs influence each others expression and how this affects the regulation of global gene expression programs we have focused on three different aspects of these ARE-BP networks: control, response to stimuli, and global effects.

To address of network control of ARE-BPs we have focused on how HuR regulates a network of mRNAs including TTP, BRF1 and HuR's own mRNA. We demonstrate that HuR can bind to TTP's, BRF1's and its own mRNA. Furthermore, by employing overexpression and siRNA knockdown approaches we demonstrate that these mRNAs and their corresponding 3'UTR luciferase reporters are resilient to fluctuations in HuR levels and that the degree of this resiliency is cell type and condition specific.

To address the temporal responses within an ARE-BP network we focused on how each of the members of the TTP family of ARE-BPs reacts following the induction of the other family members by using epidermal growth factor (EGF) stimulation. Here we show that induction of TTP family member mRNAs during EGF stimulation is partially attributable to changes in mRNA stability. Furthermore, we also show that TTP and BRF1 are able to bind each of the TTP family member mRNAs and subsequently affect their expression by altering their mRNA degradation rates. In addition, we demonstrate that the unique temporal induction patterns of the TTP family member RBPs is correlated with the EGF stimulated induction of TTP-bound mRNAs, suggesting that a network comprised of TTP family members is able to influence the timing of complex gene expression patterns.

Finally, to address the influence of these networks on regulation of global gene expression programs we have focused on how HuR recognizes AREs and whether it can globally recognize multiple classes of ARE-containing mRNAs, including the canonical class of AREs recognized by the TTP family members. To investigate how the three RNA recognition motifs (RRMs) of HuR contribute to ARE recognition we generated a series of RRM point mutants and test their ability to disrupt RNA recognition of each of the RRMs. To identify different classes of ARE-containing mRNAs we examined these mutants with a global RNA binding site detection method called photoactivatable ribonucleoside crosslinking immunoprecipitation (PAR-CLIP). Together these techniques suggest that the RRMs of HuR cooperate to recognize mRNA targets and that HuR's ability to bind RNA is coupled to the cellular distribution of HuR, and thus, are important in its role for regulating expression of bound mRNAs.

Together these studies indicate that ARE-BP posttranscriptional networks are highly interconnected and display complex regulatory interactions depending on cell type and stimuli. Furthermore, these networks can create complex behaviors such as timing of expression events or resiliency to fluctuations in protein levels. Finally, the components of these ARE-BP networks target partially overlapping sets of mRNAs to impact global gene expression patterns that ultimately coordinate the cellular responses to external stimuli.