p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.     

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

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.     

The yeast poly(A)-binding protein Pab1p stimulates in vitro poly(A)-dependent and cap-dependent translation by distinct mechanisms.

Translation initiation in extracts from Saccharomyces cerevisiae involves the concerted action of the cap-binding protein eIF4E and the poly(A) tail-binding protein Pab1p. These two proteins bind to translation initiation factor eIF4G and are needed for the translation of capped or polyadenylated mRNA, respectively. Together, these proteins synergistically activate the translation of a capped and polyadenylated mRNA. We have discovered that excess Pab1p also stimulates the translation of capped mRNA in extracts, a phenomenon that we define as trans-activation. Each of the above activities of Pab1p requires its second RNA recognition motif (RRM2). We have found that RRM2 from human PABP cannot substitute functionally for yeast RRM2. Using the differences between human and yeast RRM2 sequences as a guide, we have mutagenized yeast RRM2 and discovered residues that are required for eIF4G binding and poly(A)-dependent translation but not for trans-activation. Similarly, other residues within RRM2 were found to be required for trans-activation but not for eIF4G binding or poly(A)-dependent translation. These data show that Pab1p has at least two biochemically distinct activities in translation extracts.     

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.     

The RNA recognition motif of yeast translation initiation factor Tif3/eIF4B is required but not sufficient for RNA strand-exchange and translational activity.

The Saccharomyces cerevisiae TIF3 gene encodes a 436-amino acid (aa) protein that is the yeast homologue of mammalian translation Initiation factor eIF4B. Tif3p can be divided into three parts, the N-terminal region with an RNA recognition motif (RRM) (aa 1-182), followed in the middle part by a sevenfold repeat of 26 amino acids rich in basic and acidic residues (as 183-350), and a C-terminal region without homology to any known sequence (aa 351-436). We have analyzed several Tif3 proteins with deletions at their N and C termini for their ability (1) to complement a tif3delta strain in vivo, (2) to stimulate Tif3-dependent translation extracts, (3) to bind to single-stranded RNA, and (4) to catalyze RNA strand-exchange in vitro. Here we report that yeast Tif3/eIF4B contains at least two RNA binding domains able to bind to single-stranded RNA. One is located in the N-terminal region of the protein carrying the RRM, the other in the C-terminal two-thirds region of Tif3p. The RRM-containing domain and three of the seven repeat motifs are essential for RNA strand-exchange activity of Tif3p and translation in vitro and for complementation of a tif3delta strain, suggesting an important role for RNA strand-exchange activity in translation.     

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.     

Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR.

TIA-1 and TIAR are RNA binding proteins of the RNA recognition motif (RRM)/ribonucleoprotein (RNP) family that have been implicated as effectors of apoptotic cell death. We report the structures of murine TIA-1 and TIAR (mTIA-1 and mTIAR) deduced from cDNA cloning, the mRNA and protein tissue distribution of mTIA-1 and mTIAR, and the exon-intron structures of the mTIA-1 and mTIAR genes. Both mTIA-1 and mTIAR are comprised of three approximately 100 amino acid N-terminal RRM domains and a approximately 90 amino acid C-terminal auxiliary domain. This subfamily of RRM proteins is evolutionarily well conserved; mTIA-1 and mTIAR are 80% similar to each other, and 96 and 99% similar to hTIA-1 and hTIAR, respectively. The overall exon-intron structures of the mTIA-1 and mTIAR genes are also similar to each other, as well as to the human TIA-1 gene structure. While Northern blot analysis reveals that mTIA-1 and mTIAR mRNAs have a broad tissue distribution, mTIA-1 and mTIAR proteins are predominantly expressed in brain, testis and spleen. At least two isoforms of both mTIA-1 and mTIAR are generated by alternative splicing. Murine TIA-1 isoforms including or lacking the exon 5 encoded sequences are expressed at a ratio of approximately 1:1, whereas mTIAR isoforms including or lacking the 5'-end of exon 3 sequences are expressed in a approximately 1:6 ratio. Molecular characterization of murine TIA-1 and TIAR RNA binding proteins provides the basis for a genetic analysis of the functional roles of these proteins during mammalian development.     

A structural insight into the C-terminal RNA recognition motifs of T-cell intracellular antigen-1 protein

T-cell intracellular antigen-1 (TIA-1) plays a pleiotropic role in cell homeostasis through the regulation of alternative pre-mRNA splicing and mRNA translation by recognising uridine-rich sequences of RNAs. TIA-1 contains three RNA recognition motifs (RRMs) and a glutamine-rich domain. Here, we characterise its C-terminal RRM2 and RRM3 domains. Notably, RRM3 contains an extra novel N-terminal α-helix (α(1)) which protects its single tryptophan from the solvent exposure, even in the two-domain RRM23 context. The α(1) hardly affects the thermal stability of RRM3. On the contrary, RRM2 destabilises RRM3, indicating that both modules are tumbling together, which may influence the RNA binding activity of TIA-1.     

Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP

The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF(65) complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C. Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3. It inserts a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs.     

Solution structure of the second RNA recognition motif (RRM) domain of murine T cell intracellular antigen-1 (TIA-1) and its RNA recognition mode

T cell intracellular antigen-1 (TIA-1), an apoptosis promoting factor, functions as a splicing regulator for the Fas pre-mRNA. TIA-1 possesses three RNA recognition motifs (RRMs) and a glutamine-rich domain. The second RRM (RRM2) is necessary and sufficient for tight, sequence-specific binding to the uridine-rich sequences buried around the 5' splice sites. In the present study, we solved the solution structure of the murine TIA-1 RRM2 by heteronuclear-nuclear magnetic resonance spectroscopy. The TIA-1 RRM2 adopts the RRM fold (betaalphabetabetaalphabeta) and possesses an extra beta-strand between beta2 and beta3, which forms an additional beta-sheet with the C-terminal part of beta2. We refer to this structure as the beta2-beta2' beta-loop. Interestingly, this characteristic beta-loop structure is conserved among a number of RRMs, including the U2AF65 RRM2 and the Sex-lethal RRM1 and RRM2, which also bind to uridine-rich RNAs. Furthermore, we identified a new sequence motif in the beta2-beta2' beta-loop, the DxxT motif. Chemical shift perturbation analyses of both the main and side chains upon binding to the uridine pentamer RNA revealed that most of the beta-sheet surface, including the beta2-beta2' beta-loop, is involved in the RNA binding. An investigation of the chemical shift perturbation revealed similarity in the RNA recognition modes between the TIA-1 and U2AF65 RRMs.     

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre-mRNA 5' splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5' ss is promoted by binding of the protein TIA-1 to uridine-rich sequences immediately downstream from the 5' ss. Here we describe a molecular dissection of the activities of TIA-1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre-mRNA. The non- consensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5' ss. Co-precipitation experiments revealed a specific and direct interaction involving the N-terminal region of the U1 protein U1-C and the Q-rich domain of TIA-1, an interaction enhanced by RRM1. The results argue that binding of TIA-1 in the vicinity of a 5' ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1-C, thus providing one molecular mechanism for the function of this splicing regulator.     

RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.     

The C-Terminal Domain of SRA1p Has a Fold More Similar to PRP18 than to an RRM and Does Not Directly Bind to the SRA1 RNA STR7 Region

Steroid receptor activator RNA protein (SRA1p) is the translation product of the bi-functional long non-coding RNA steroid receptor activator RNA 1 (SRA1) that is part of the steroid receptor coactivator-1 acetyltransferase complex and is indicated to be an epigenetic regulatory component. Previously, the SRA1p protein was suggested to contain an RNA recognition motif (RRM) domain. We have determined the solution structure of the C-terminal domain of human SRA1p by NMR spectroscopy. Our structure along with sequence comparisons among SRA1p orthologs and against authentic RRM proteins indicates that it is not an RRM domain but rather an all-helical protein with a fold more similar to the PRP18 splicing factor. NMR spectroscopy on the full SRA1p protein suggests that this structure is relevant to the native full-length context. Furthermore, molecular modeling indicates that this fold is well conserved among vertebrates. Amino acid variations in this protein seen across sequenced human genomes, including those in tumor cells, indicate that mutations that disrupt the fold occur vary rarely and highlight that its function is well conserved. SRA1p had previously been suggested to bind to the SRA1 RNA, but NMR spectra of SRA1p in the presence of its 80-nt RNA target suggest otherwise and indicate that this protein must be part of a multi-protein complex in order to recognize its proposed RNA recognition element.     

Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95 Å resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.     

The role of the C-terminal helix of U1A protein in the interaction with U1hpII RNA

Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a ‘lure and lock’ model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA–protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.     

Spliceosomal SL1 RNA binding to U1-70K: the role of the extended RRM

The RNA recognition motif (RRM) occurs widely in RNA-binding proteins, but does not always by itself support full binding. For example, it is known that binding of SL1 RNA to the protein U1-70K in the U1 spliceosomal particle is reduced when a region flanking the RRM is truncated. How the RRM flanking regions that together with the RRM make up an ‘extended RRM’ (eRRM) contribute to complex stability and structural organization is unknown. We study the U1-70K eRRM bound to SL1 RNA by thermal dissociation and laser temperature jump kinetics; long-time molecular dynamics simulations interpret the experiments with atomistic resolution. Truncation of the helix flanking the RRM on its N-terminal side, ‘N-helix,’ strongly reduces overall binding, which is further weakened under higher salt and temperature conditions. Truncating the disordered region flanking the RRM on the C-terminal side, ‘C-IDR’, affects the local binding site. Surprisingly, all-atom simulations show that protein truncation enhances base stacking interactions in the binding site and leaves the overall number of hydrogen bonds intact. Instead, the flanking regions of the eRRM act in a distributed fashion via collective interactions with the RNA when external stresses such as temperature or high salt mimicking osmotic imbalance are applied.     

The N-terminal RNA Recognition Motif of PfSR1 Confers Semi-specificity for Pyrimidines during RNA Recognition

Alternative splicing confers a complexity to the mRNA landscape of apicomplexans, resulting in a high proteomic diversity. The Plasmodium falciparum Ser/Arg-rich protein 1 (PfSR1) is the first protein to be confirmed as an alternative splicing factor in this class of parasitic protists [1]. A recent study [2] showed a purine bias in RNA binding among cognate RNA substrates of PfSR1. Here, we have investigated the role played by the amino-terminal RNA recognition motif (RRM1) of PfSR1 from the solution structure of its complex with ACAUCA RNA hexamer to understand how its mechanism of RNA recognition compares to human orthologs and to the C-terminal RRM. RNA binding by RRM1 is mediated through specific recognition of a cytosine base situated 5′ of one or more pyrimidine bases by a conserved tyrosine residue on β₁ and a glutamate residue on the β₄ strand. Affinity is conferred through insertion of a 3′ pyrimidine into a positively charged pocket. Retention of fast dynamics and ITC binding constants indicate the complex to be of moderate affinity. Using calorimetry and mapping of NMR chemical shift perturbations, we have also ascertained the purine preference of PfSR1 to be a property of the carboxy terminal pseudo-RRM (RRM2), which binds RNA non-canonically and with greater affinity compared to RRM1. Our findings show conclusive evidence of complementary RNA sequence recognition by the two RRMs, which may potentially aid PfSR1 in binding RNA with a high sequence specificity.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon

Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids.     

Cell proteins TIA-1 and TIAR interact with the 3' stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication

It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3'-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3'(-) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3'(-) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3'(-) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3'(-) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (K(d)) for the interaction between TIAR RRM2 and the WNV 3'(-) SL RNA was 1.5 x 10(-8), while that for TIA-1 RRM2 was 1.12 x 10(-7). WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.