Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR

Mammalian gene expression is extensively regulated at the posttranscriptional level, via mechanisms such as pre-mRNA splicing, transport, stability, and translation. Prominent among the posttranscriptional trans-acting factors that influence these processes are RNA-binding proteins (RBPs) that influence transcript splicing, localization, stability, and association with the translation machinery (11, 17, 42). Many mRNAs encoding stress-response, proliferative, immune, and developmental proteins comprise specific regulatory sequences in the untranslated regions (UTRs), often encompassing uridine- or adenine/uridine-rich stretches (hence termed “AREs”). AREs are bound by a specific subset of RBPs that influence the stability and translation of the ribonucleoprotein (RNP) complex. Many ARE-RBPs decrease the stability of target mRNAs, including AU-binding factor 1 (AUF1), tristetraprolin (TTP), K homology splicing-regulatory protein (KSRP), and the butyrate response factor 1 (BRF1) (7, 27, 34, 39, 46). Other ARE-RBPs, like the Hu proteins (HuR, HuB, HuC, and HuD), can stabilize target mRNAs instead (4, 6); Hu proteins have also been shown to modulate the translation of several target mRNAs, both enhancing (5, 24, 31) and inhibiting (9, 23, 33) protein synthesis. However, the best-studied ARE-RBPs functioning as translational inhibitors are the T-cell-restricted intracellular antigen 1 (TIA-1) and the TIA-1-related protein TIAR (1, 2, 14, 28, 32). TIA-1 and TIAR contain three RNA-recognition motifs (RRMs) through which they bind mRNAs (10). In addition to participating in pre-mRNA splicing (12, 26, 38), TIA-1 and TIAR have been proposed to repress translation (1, 2, 32, 37). In unstressed cells, a preinitiation complex (comprising the eukaryotic translation initiation factor 1 [eIF-1], eIF-2, eIF-3, eIF-5, and the 40S ribosomal subunit) forms at the 5′ end of capped mRNAs. Following the recognition of an initiation codon, the 60S subunit assembles, displacing the eIFs and forming a functional ribosome to initiate translation. In cells exposed to damaging agents, phosphorylation of eIF-2α by a family of kinases (PKR, PERK, GCN2, and HRI) reduces the levels of functional preinitiation complex (recently reviewed in reference 16). Under these conditions, TIAR and TIA-1 have been postulated to function as translational repressors by associating with eIF-4F, eIF-3, and the 40S ribosomal subunit, to form nonfunctional preinitiation complexes (2). The self-aggregating properties of TIA-1 and TIAR were further proposed to facilitate the accumulation of the translationally inactive preinitiation complexes into discrete cytoplasmic foci called stress granules (SGs). Given the presence of RBPs implicated in the regulation of mRNA turnover (such as TTP and HuR) and translation (TIA proteins) at SGs, these foci are believed to function as dynamic sites of mRNA triage during stress, wherein the composition of mRNA RNP complexes and their subsequent engagement with the translation or degradation machineries are decided (20, 21). While these mechanisms of TIA-1/TIAR action can lead to a general suppression of translation in the cell, they are believed to have a preferential effect upon specific subsets of bound mRNAs, such as ARE-containing mRNAs encoding tumor necrosis factor alpha (TNF-α), matrix metalloproteinase 13 (MMP-13), cyclooxygenase 2 (COX-2), and β2-adrenergic receptor (AR) (8, 14, 19, 37, 44). Accordingly, global searches have been undertaken to identify TIA-1/TIAR target mRNAs systematically. An earlier study in which pools of random RNA sequences were selected/amplified in vitro revealed that both TIA proteins recognized RNAs containing U-rich stretches (10). More recently, a genome-wide search for TIA-1 target mRNAs was carried out by immunoprecipitation (IP) of TIA-1 RNPs followed by the identification of bound transcripts using DNA microarrays (28). A computational analysis of the target mRNAs found by this approach led to the elucidation of a shared signature motif present among mRNAs which was also U rich. TIA-1 was shown to associate with target mRNAs bearing this motif in cells subjected to heat shock and to suppress their translation (28). Using a similar en masse approach, TIAR target mRNAs were found to include many mRNAs encoding translation factors and other proteins involved in translation (32). Further analysis of TIAR RNPs indicated that the association of TIAR with several target mRNAs increased following irradiation with short-wavelength UV light (UVC), thereby helping to suppress their translation (32). Here, we sought to elucidate a shared motif among TIAR target mRNAs. The starting material was a collection of TIAR-bound transcripts that was isolated from untreated RKO cells (a human colon cancer line) using the RNP IP methodology and was identified by using a microarray (32). Computational analysis of this set of transcripts revealed a shared signature motif that was unexpectedly C rich. The ability of TIAR RRM domains to bind to a representative C-rich sequence was verified in vitro using surface plasmon resonance (SPR). Further validation of this interaction was obtained by studying the binding of mRNAs that were predicted to be TIAR targets because their 3′ untranslated regions (3′UTRs) contained at least one occurrence of the C-rich TIAR motif. Interestingly, the transcripts tested were found to dissociate from TIAR in response to UVC treatment, suggesting that this C-rich TIAR signature motif may occur within mRNAs whose binding to TIAR decreases following stress stimulation.