Journal of Molecular Biology
Volume 415, Issue 4, 27 January 2012, Pages 727-740
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Three RNA Recognition Motifs Participate in RNA Recognition and Structural Organization by the Pro-Apoptotic Factor TIA-1

https://doi.org/10.1016/j.jmb.2011.11.040Get rights and content

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.

Graphical Abstract

Highlights

► Three tandem RRMs are required for TIA-1 to bind a polyU RNA with high affinity. ► The N-terminal RRM is not required for TIA-1 to bind a fas splice site RNA. ► The average solution conformation of three TIA-1 RRMs is an obtuse V shape. ► The tandem TIA-1 RRMs become more compact when bound to RNA.

Introduction

The T-cell intracellular antigen-1 (TIA-1) and the TIA-1-related (TIAR) proteins are essential; thus, mice lacking TIA-1 or TIAR show high rates of embryonic lethality.1, 2 TIA-1 and TIAR play dual roles in promoting pre-mRNA splicing and repressing mRNA translation. In its capacity as a pre-mRNA splicing factor, TIA-1 activates the 5′ splice sites of transcripts including those encoding the fas apoptosis-promoting receptor,3 cystic fibrosis transmembrane conductance regulator,4 and Survival Motor Neuron 2,5 among others. To accomplish its nuclear function in pre-mRNA splicing, TIA-1 binds U-rich RNA sites located downstream of weak 5′ splice sites (Fig. 1a). There, TIA-1 engages the U1 small nuclear ribonucleoprotein (snRNP) component of the spliceosome via protein–protein interactions with its U1-C subunit.6, 7 As an alternative to recruiting the U1 snRNP, TIAR has been found to enhance association of the U6 snRNP with pre-mRNA sites.8, 9 In the cytoplasm, TIA-1 and TIAR regulate translation of various mRNAs including tnf-α and cox-2 mRNAs that are involved in apoptotic pathways and can be defective in inflammation and arthritis.1, 10, 11 In this capacity, TIA-1 typically represses translation by binding 3′ untranslated regions (UTRs) marked by class II AU-rich elements, a series of partially overlapping AUUUA pentamers within a uridine-rich context (Fig. 1a).12 The mechanism of TIA-1-induced translational repression has been characterized for conditions of environmental stress, such as oxidation, heat, or starvation (reviewed in Refs. 13 and 14). Under these conditions, eIF2α phosphorylation inhibits translation initiation, and TIA-1 or TIAR escorts the unproductive translation initiation complexes with the mRNA to stress granules.15, 16, 17

Although TIA-1 and TIAR functions are becoming better understood, much remains to be learned concerning their underlying molecular actions. The TIA-1 and TIAR proteins share 80% sequence identity and similar domain organizations of three RNA recognition motifs (RRMs) and a C-terminal Q-rich domain (Fig. 1b).18, 19 The Q-rich domain acts as a protein–protein interaction motif to promote U1-C interactions or TIA-1 aggregation and does not contribute to the RNA affinity of TIA-1.7, 20 Instead, attention on the region responsible for recognizing the U-rich elements of splice sites and 3′ UTRs has focused on the RRMs of TIA-1 or TIAR. Structures of individual TIA-1 or TIAR RRMs (RIKEN Structural Genomics Initiative and Refs. 21 and 22) establish that all three RRMs possess a canonical αβ fold prevalent among this class of RNA binding domain.23 The TIA-1 or TIAR RRMs display consensus ribonucleoprotein (RNP1 and RNP2) residues at the expected positions for RNA binding (Fig. 1c). Indeed, the importance of the central RRM2 of TIA-1 for RNA recognition is well established, since the isolated RRM2 domain binds the msl-2 5′ splice site region,7 viral RNAs,24 and SELEX RNAs25 in gel shift experiments. The C-terminal TIA-1 RRM3 also binds RNA, since the isolated RRM3 domain of TIA-1 or TIAR affinity-precipitates cellular RNAs.25 In contrast, the role of the N-terminal RRM1 of TIA-1 or TIAR is less clear. In the absence of the other domains, the isolated TIA-1 or TIAR RRM1 lacks detectable RNA binding,7, 25, 26 although the isolated TIAR RRM1 binds DNA.26 Instead, some reports suggest that TIA-1 RRM1 primarily assists a C-terminal Q-rich domain of TIA-1 to interact with the U1 snRNP-C (U1-C) subunit and hence stabilizes U1 snRNP association with regulated splice sites.6, 7

Here, we used isothermal titration calorimetry (ITC) to investigate the role of the TIA-1 RRMs in RNA recognition and complementary small-angle X-ray scattering (SAXS) to determine the solution shape of the TIA-1-RRM-containing domain in the presence and absence of RNA. ITC characterization of site-directed mutant proteins demonstrates that all three RRMs are required for high-affinity recognition of polyU RNAs but that RRM1 is dispensable for lower-affinity binding to a natural fas pre-mRNA site. The SAXS analyses indicate that TIA-1 undergoes a conformational change in the RNA-bound state. Ab initio models of TIA-1 reveal an obtuse “V” shape positioning the amino- and carboxy-termini on the same face of the molecule, a result that has important implications for U1 snRNP recruitment to the 5′ splice site.

Section snippets

Two identical sites for TIA-1 binding a fas RNA target

We used ITC to investigate the affinity, enthalpy, and entropy changes for TIA-1 binding a target site from the fas pre-mRNA (Fig. 2 and Table 1). A nearly full length TIA-1 construct containing three RRMs (wtTIA-RRM123, residues 1–274) (Fig. 1b) was titrated into a 31-nucleotide AU-rich RNA derived from the 5′ splice site of fas exon 5 (fas RNA, Fig. 1a). Two identical binding sites for wtTIA-RRM123 were observed in the fas RNA isotherm (〈χ〉2 5.7E5 for identical site versus 〈χ〉2 2.5E5 for

Discussion

RRMs are among the most prevalent RNA interaction motifs of eukaryotic proteins (reviewed in Ref. 23). The structures of canonical RRM-containing proteins share salient features, and conserved aromatic RNP residues of the β-strands generally stack with the RNA bases in a consistent 5′-to-3′ direction of the RNA strand relative to the RRM fold. We resolved distinct binding sites using ITC, a technique with sufficient sampling, and investigated the contributions of TIA-1 RRMs to RNA recognition

Protein expression and purification

Human wtTIA-RRM123 (residues 1–274) was subcloned from full-length TIA-1 (National Center for Biotechnology Information Refseq NP_071320) into pGEX-6p-2. Double mutations Y10A/F50A, F98A/F140A, and Y206A/F242A, respectively, generated the TIA-Mut1, Mut2, and Mut3 constructs. The glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli BL21 Rosetta-2 cells (Merck Novagen) and purified by glutathione affinity chromatography, and the GST tag was cleaved overnight. A

Acknowledgements

J. Valcárcel graciously shared the TIA-1 cDNA as the basis for expression plasmid construction. We thank M. Swenson and A. O. Kumar for preliminary ITC experiments; Drs. G. L. Hura and R. Gillilan for assistance with SAXS data collection; and Drs. J. E. Wedekind, J. F. Kielkopf, and M. Sattler for insightful discussions. This work was funded by a grant from the National Institutes of Health (R01 GM070503) to C.L.K. SAXS data collected at the SIBYLS beamline of the ALS, Lawrence Berkeley

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