Article

Three RNA Recognition Motifs Participate in RNA Recognition and Structural Organization by the Pro-Apoptotic Factor TIA-1

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 12/2011; 415(4):727-40. DOI: 10.1016/j.jmb.2011.11.040
Source: PubMed

ABSTRACT T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.

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    • "This is the first demonstration that this splice variant may be present in human cancers, and that it may regulate VEGF splice form expression. sTIA-1, containing only one RNA recognition motif (RRM), prevented binding of full length TIA-1 to VEGF-A 165 mRNA, blocking both its angiogenic VEGF splice site repression and its promotion of translation of anti-angiogenic VEGF-A 165 b. It has previously been shown that RRM2 and 3 are required for binding of TIA-1 to RNA (Bauer et al., 2012), and interestingly, RRM3 is required for binding to C rich stretches (Cruz-Gallardo et al., 2014). Moreover, inclusion of exon 5 in TIA-1 results in a higher splice activity, indicating that the activity of the protein can be altered by alternative splicing (Izquierdo and Valcarcel, 2007). "
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    • "It is thus likely that a main function of RRM1 is to mediate protein–protein rather than RNA interactions. Consistent with this notion is the recent observation that RRM1 is dispensable for binding to an AU-rich RNA ligand (24). Some minor contribution to binding of poly-U RNA reported by the same authors may indeed reflect non-specific RRM1 interactions, consistent with our NMR titrations. "
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    • "TIA-1 has been described as an ARE-binding protein and binds both U-rich and AU-rich elements. The preference for U-rich regions was shown using SELEX [47], cross-linking and immunoprecipitation [48] and isothermal titration calorimetry (ITC) [49]. Just recently, the high affinity toward binding to U-rich RNA could be traced to six amino acid residues in the TIA1 RNA recognition motif 2 (RRM2)[50]. "
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