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Biochemical and Biophysical Research Communications 367 (2008) 813–819

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

Amit O. Kumar a,1, Matthew C. Swenson b,1, Matthew M. Benning c, Clara L. Kielkopf a,*

a Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USAb Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA

c Bruker AXS Inc., 5465 East Cheryl Parkway, Madison, WI 53711, USA

Received 28 December 2007Available online 15 January 2008

Abstract

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in thecytoplasm, 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 A resolution. Comparison withstructurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1–RRM2. The ver-satile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein–protein interactionsmediated by the RNP motifs of TIA-1–RRM2. Importantly, the TIA-1–RRM2 structure reveals the locations of mutations responsiblefor inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior ofthe domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.� 2008 Elsevier Inc. All rights reserved.

Keywords: RRM; RNA binding domain; Pre-mRNA splicing; mRNA translation; TIA-1; TIAR

Post-transcriptional mechanisms for regulating geneexpression depend on numerous coordinated interactionsamong RNA sequences and trans-acting protein factors.The RNA binding protein TIA-1 (T-cell-restricted intracel-lular antigen-1) functions as a post-transcriptional regula-tor of gene expression by recognizing uridine-rich RNAsites. In the nucleus, TIA-1 regulates alternative splicingof pre-mRNAs, including those encoding fibroblast growthfactor receptor-2 (fgfr-2), type II procollagen (col2a1), thecystic fibrosis transmembrane conductance regulator (cftr),and the pro-apoptotic Fas receptor (fas), among others[1–6]. In the cytoplasm, TIA-1 regulates mRNA translation[7–10], and suppresses mRNA translation during environ-mental stress (reviewed in [11]). The functional importanceof TIA-1 is shown by its requirement for viability of DT40

0006-291X/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2008.01.027

* Corresponding author. Fax: +1 585 275 6007.E-mail address: clara_kielkopf@urmc.rochester.edu (C.L. Kielkopf).

1 These authors contributed equally to this work.

cells [12], and the high rates of embryonic lethality amongmice lacking TIA-1 [8,13].

The TIA-1 polypeptide contains three RNA recognitionmotifs (RRMs, a prevalent class of RNA binding domain)and a C-terminal, Q-rich domain (Fig. 1) [14,15]. Inhumans, a second gene encodes the nearly identical TIARprotein [16], and at least two alternative spliceoforms existfor both TIA-1 and TIAR [14,15,17]. The Q-rich effecterdomain directs TIA-1-associated mRNAs to cytoplasmicstress granules during translational silencing [18], andrecruits the U1 spliceosomal component (snRNP) to weak50 splice sites during nuclear pre-mRNA splicing [19]. Likethe Q-rich domain, the N-terminal RRM (RRM1) partici-pates in U1 snRNP recruitment [19], and additionallyassociates with single-stranded DNA molecules [20]. TheC-terminal RRM (RRM3) co-precipitates with cellularRNAs, however interactions of TIA-1–RRM3 with uri-dine-rich RNAs are not detected in vitro [21]. The centralRRM (RRM2) of TIA-1 is the established, uridine-specific

H. sapiens HFHVFVGDLSPEITTEDIKAAFA-PFGRISDARVVKDMATGKSKGYGFVSFFNKWDAENAIQQMGGQWLGGRQIRTNWATM. musculus HFHVFVGDLSPEITTEDIKAAFA-PFGRISDARVVKDMATGKSKGYGFVSFFNKWDAENAIQQMGGQWLGGRQIRTNWATG. gallus HFHVFVGDLSPEITTEDIKAAFA-PFGRISDARVVKDMATGKSKGYGFVSFFNKWDAENAIQQMGGQWLGGRQIRTNWATC. elegans HFHVFVGDLSSEVDNQKLREAFQ-PFGDVSDAKVIRDTNTTKSKGYGFVSYPKREEAERAIEQMNGQWLGRRTIRTNWATN. crassa HFHIFVGDLSNEVNDEVLLQAFS-AFGSVSEARVMWDMKTGRSRGYGFVAFRDRPDAEKALSSMDGEWLGSRAIRCNWANA. thaliana HFNIFVGDLSPEVTDATLYQSFS-VFSSCSDARVMWDQKTGRSRGFGFVSFRNQQDAQTAINEMNGKWLSSRQIRCNWATB. mori HHHIFVGDLSPEIETNILREAFA-PFGEISNCRIVRDPQTLKSKGYAFVSFVKKADAEAAIQAMNGQWLGSRSIRTNWSTE. histolytica NYKIFVGGLQPEVNDDLLYKTFQ-KFGRVTDARVLKFTQTGKSKGYGFVTFIRKEDAETAMQMMNGEKLEGRNIKVNWVTS. cerevisiae NCSIFVGDLAPNVTESQLFELFINRYASTSHAKIVHDQVTGMSKGYGFVKFTNSDEQQLALSEMQGVFLNGRAIKVGPTS

RNP2 RNP1

human TIA-1

7

α1 β2 β3 α 2 β4 3101 β1 β5

N C RRM2 Q rich RRM1 RRM3

81 94 172 202 274 284 374

Fig. 1. Domain structure of human TIA-1 above a sequence alignment of TIA-1–RRM2 from representative homologs. Sequences are colored by percentidentity: 100% = green, <90% = sea green, <80% = lime, <70% = bright green, <60% = yellow, and <50% = white. Secondary structure elements areindicated above the alignment [45]. The RNP1 and RNP2 are boxed and labeled below the alignment. Surface residues of human Tia-1 mutated tofacilitate crystallization are highlighted by blue filled circle. Residues that interact with RNA in both PAB and SXL complexes are marked with red filledcircle. Accession numbers for aligned sequences include: Homo sapiens (NP_071320), Mus musculus (NP_035715), Gallus gallus (XP_001233215),Caenorhabditis elegans (NP_495121), Neurospora crassa (XP_962723), Arabidopsis thaliana (NP_188026), Bombyx mori (NP_001036895), Entamoeba

histolytica (XP_649094), Saccharomyces cerevisiae (CAA46011).

Table 1Crystallographic data and refinement statistics

(A) Data collection statisticsa

Space group P61

Unit cella = b, c (A) 56.50, 76.70Resolution limit (A) 1.95Rsym

b(%) 7.0 (27.9)I/r (I) 15.5 (3.5)Completeness (%) 99.8 (99.8)Redundancy 5.2 (3.2)

(B) Refinement statistics

Resolution range (A) 20.0–1.95No. reflections(working/test set) 9648/489Rcryst/Rfree

c (%) 21.6/24.5No. protein atoms 1221No. water molecules 118No. iodide ions 7RMSD bond lengths (A) 0.006RMSD bond angles (�) 1.31

Average B-factors (A2)

All atoms 15.2Protein atoms 14.2Water molecules 23.2

a Values in parenthesis are for the highest resolution shell (2.04–1.95 A).b Rsym ¼

Phkl

PijI i � hIij=

Phkl

PiI i, where Ii is an intersity I for the ith

measurement of a reflection with indices hkl and hIi is the weighted meanof all measurements of I.

c Rcryst ¼P

hkl

PijjF oðhklÞj � kjF cðhklÞjj=

PhkljF oðhklÞj for the working

set of reflections; Rfree is Rcryst for 5% of the reflections excluded from therefinement.

814 A.O. Kumar et al. / Biochemical and Biophysical Research Communications 367 (2008) 813–819

region, as demonstrated by the ability of isolated TIA-1–RRM2 to bind the msl-2 50 splice site region [19], viralRNAs [22], and uridine-rich SELEX RNAs [21]. Further-more, the RRM2 region is necessary and sufficient fornuclear import of TIA-1 [23]. To investigate the majorRNA binding surface of TIA-1, we have determined thestructure of RRM2 in the absence of RNA at 2.0 Aresolution.

Materials and methods

Protein purification. The human TIA-1–RRM2 fragment (residues 94–175) was expressed using the pGEX-6p vector (GE Healthcare). Gluta-thione-S-transferase (GST) fusion proteins were purified by glutathioneaffinity, and then the TIA-1 fragment was further purified by cationexchange chromatography following removal of the GST tag. The TIARRM2 and crystallized variant (described below) were concentrated to>15 mg/mL in 100 mM NaCl, 25 mM Hepes pH 7.4, 0.2 mM TCEP.

Crystallization. Initial crystals of TIA-1–RRM2 grew in clusters of fineneedles (<25 � 200 lm) (Supp. Fig. 1A). To improve crystal quality, thesurface entropy reduction (SER) server (http://nihserver.mbi.ucla.edu/SER) was used to identify suitable modifications of TIA-1–RRM2 topromote crystallization. The site furthest from the putative RNA bindingsurface was selected for mutagenesis. Improved crystals of the resultingSER variant (E109A-D110A-K112A) grew in 2–3 days by thehanging drop vapor diffusion method at 22 �C, from a 1:1 mixture ofprotein with 15% polyethylene glycol 3350, 0.5 M KI, 0.1 M Tris pH 8.0(Supp. Fig. 1B).

Data collection, structure determination, and refinement. X-ray datawere collected using a MICROSTAR X-ray source with X8 Proteumdetector (Bruker-AXS) (Table 1). A homology model of TIA-1–RRM2[24] based on the N-terminal RRM of poly-A binding protein (PDB-code = 1CVJ) [25] was used for molecular replacement in the programPhaser [26,27]. Refinement and model building were, respectively, com-pleted using CNS [28] and Coot [29,30]. The coordinates and structurefactors are available (PDB-code=3BS9).

Fluorescence measurements. The affinities of the wild-type RRM2 andSER-RRM2 domains of TIA-1 were measured for a 30 fluorescein-labeled20-uridine RNA (U20) (Dharmacon Research). Proteins were titrated intoa thermostatted, 0.5 cm quartz cuvette containing U20 in 100 mM NaCl,

25 mM Hepes pH 7.4 at 25 �C, and the fluorescence anisotropy valueswere measured using a Fluoromax-3 fluorimeter (Jobin-Yvon). Excitationand emission wavelengths were typically 490/520 nm, respectively. Datawere fit by non-linear regression assuming single site binding (Fig. 2) toobtain the apparent KD with the following equation, where x is the total

Fig. 2. Overall structure of TIA-1–RRM2. (A) Ribbon diagram of the two TIA-1–RRM2 molecules in the asymmetric unit of the crystal. Molecules ‘A’and ‘B’ are distinguished by subscripts of the residues labels. Secondary structure elements of molecule ‘A’ are labeled. Composite omit electron density(light blue) at 1r contour level surrounds key aromatic residues of the RNP motifs. Anomalous difference Fourier maps (magenta) at 3r contour levelindicate the iodide positions. (B) Superposition of the top five related structures identified using DALI [46]: TIA-1–RRM2 (orange), PAB (PDB-code=1CVJ, cyan), SXL (PDB-code=1B7F, magenta), hnRNP A1 (PDB-code=1HA1, green), PRP24 (PDB-code=2GHP, marine), and nuclear capbinding protein (PDB-code=1H6K, red). (C) Noncrystallographic contacts mediated by the SER mutations, viewed 180� about the y-axis relative to (A).Ball-and-stick diagrams are shown for SER residues (green) and other residues of the iodide binding sites (orange). The iodides bound at the interface areshown as magenta spheres, and the water molecules directly coordinated to these iodides are shown as blue spheres. (D) RNA binding by TIA-1–RRMand SER TIA-1–RRM2 compared using fluorescence anisotropy. Overlay of nonlinear least square fits of TIA-1–RRM2 (orange) and SER TIA-1–RRM2(green) binding 50 fluorescein-labeled 20 mer polyuridine. The average apparent equilibrium dissociation constants (KD) and standard deviations of 2–3experiments are inset.

A.O. Kumar et al. / Biochemical and Biophysical Research Communications 367 (2008) 813–819 815

protein r¼ rFþ rB�rF

2½RNA� ððKdþxþ½RNA�Þ�ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðKdþxþ½RNA�Þ2�4½RNA�x

qÞ

protein concentration, [RNA] is the total RNA concentration, r is theobserved anisotropy at the ith titration, rB is the anisotropy at zero proteinconcentration, and rF is the anisotropy at saturating protein concentration(floated in fit).

Results and discussion

Overall structure and comparison with other RRMs

The structure of TIA-1–RRM2, with a SER modifica-tion to improve crystal quality (Glu109Ala/Asp110Ala/Lys112Ala), was determined at 1.95 A resolution (Table1). Two, nearly identical TIA-1–RRM2 molecules (residues94–172, 0.4A RMSD between 79 Ca atoms) in the crystal-lographic asymmetric unit adopt canonical RRM folds(babbab topology) (Fig. 2A). Accordingly, the TIA-1–RRM2 structure most closely matches the N-terminal

RRMs of the RNA binding proteins poly-A bindingprotein (PAB) ([25]; PDB-code=1CVJ; Z-score = 15.5;RMSD = 1.1 A and 37% sequence identity for residues11–88) and sex-lethal (SXL) ([31]; PDB-code=1B7F; Z-score = 14.6; RMSD = 1.3 A and 28% identity for residues125–202) (Fig. 2B). Other close matches identified using theDALI protein structure server include hnRNP A1 ([32];PDB-code=1HA1; Z-score = 13.7; RMSD = 1.3 A and27% identity for residues 13–89), nuclear cap bindingprotein ([33]; PDB-code=1H6K; Z-score = 12.7;RMSD = 1.3 A and 34% identity for residues 40–117),and PRP24 ([34]; PDB-code=2GHP; Z-score = 12.7;RMSD = 2.0A and 21% identity for residues 41–115).The b2–b3 loop conformations of TIA-1–RRM2, PRP24,and the nuclear cap binding protein are the most variableregions of the structures, whereas b2–b3 loop conforma-tions are very similar among the structures of TIA-1–RRM2 and the other single-stranded RNA binding

816 A.O. Kumar et al. / Biochemical and Biophysical Research Communications 367 (2008) 813–819

proteins PAB, SXL, and hnRNP A1. The b2–b3 loopdifferences are likely to result from interdomain interac-tions mediated by the PRP24 b2–b3 loop, and removal ofthe nuclear cap binding protein b2–b3 loop by limited pro-teolysis. All of the aligned structures share a common 310-helical extension of the first a-helix, and a b-hairpin (b4–b5)insertion prior to the terminal b-strand (Fig. 2B).

3.2 Influence of the SER mutations on RNA binding and

crystallization

As predicted based on homology with other RRMs, theSER-modified residues are located on the exterior of thefirst a-helix, distant from the putative RNA binding sur-

Fig. 3. Comparisons with the TIA-1 RNP motifs. (A) Symmetry-related interaand green. Key interacting residues are shown with ball-and-stick representatiointermolecular interaction are colored magenta. Primed italics distinguish residu(blue) complex (PDB-code 1OO0). Regions of the mago subunit outside thSuperposition of TIA-1–RRM2 with PAB residues 11–88 (PDB-code 1CVJ). (D1B7F). TIA-1 is colored yellow-orange, PAB is blue, and SXL is green. The RNy-axis relative to (A). Residues of TIA-1 (Phe98, Arg125, Asp129, Tyr138, PhAsp45, Tyr54, Tyr56, Arg83) and SXL (Ile128, Arg155, Asp159, Tyr168, Phe

face presented by the b-sheet (Fig. 2C). To ascertain thatthe SER–RRM2 binds RNA comparably to unmodifiedRRM2 of TIA-1, fluorescence anisotropy assays were usedto compare the affinities of the wild-type and SER–RRM2for uridine-rich RNA. The SER mutations had no detect-able effect on the polyuridine-binding characteristics ofTIA-1–RRM2 (Fig. 2D), demonstrating the utility of thesemutations for structural investigations of TIA-1–RNAbinding.

Rather than mediating crystallographic symmetry-related interactions, the SER residues are located at theinterface of two RRM2 molecules in the asymmetric unit(Fig. 2C). Juxtaposition of the SER alanine-replacementsfor Glu109, Asp110, and Lys112, coupled with surround-

ctions between two TIA-1–RRM2 molecules, respectively, colored yellown, and consensus residues of the RNP motifs responsible for mediating thee labels of the symmetry-related molecule. (B) View of the Y14 (red)-mago

e a-helical interactions with the Y14 RRM are omitted for clarity. (C)) Superposition of TIA-1–RRM2 with SXL residues 125–202 (PDB-code

A strands are colored magenta. The view is rotated 90� clockwise about thee140, Arg167) are labeled. Corresponding residues of PAB (Tyr14, Arg41,170, Lys197) are shown as ball-and-stick representations.

Fig. 4. Site-directed mutations used to investigate TIAR nuclear locali-zation mapped on the TIA-1–RRM2 structure. Side chains of solvent-exposed mutated residues are shown with ball-and-stick representationand colored orange. The mutated glycines are indicated by spheres.Leu102 of the RNP2 motif (blue) and Val141 of the RNP1 motif (red) areburied within the RRM interior. Residues that contact Leu102 are shownwith ball-and-stick representation, and colored salmon. Residues thatcontact Val141 are also shown, and colored light blue. Residues thatcontact both Leu102 and Val141 are colored violet.

A.O. Kumar et al. / Biochemical and Biophysical Research Communications 367 (2008) 813–819 817

ing hydrophobic residues, created a nonpolar iodide-bind-ing site. An iodide with a significant anomalous differenceFourier signal (22r) makes eight contacts ranging from3.4 A (H2O—I-) to 4.6 A (Glu109Ala methyl group—I-)in length, consistent with the experimentally determined3.6-3.7 A distances of iodide hydration [35] and coordina-tion distances of iodides bound nonspecifically to proteins[36]. Anti-parallel association of the N-terminal a-helicesabout the noncrystallographic two-fold axis places theb-sheet surfaces of the two molecules on the equivalent faceof the RRM2 dimer. Although the SER mutationspromote a noncrystallographic interaction between twoTIA-1 RRMs, this interaction in turn positions the mole-cules appropriately to build the crystal lattice.

3.3 Symmetry-related protein–protein interactions mediated

by RNP motifs

Although the level of primary sequence identity amongmost RRMs is low (<25%), two ribonucleoprotein consen-sus motifs (RNP1 and RNP2) in the central b-strands com-monly display aromatic stacking interactions with boundnucleic acids. Nevertheless, the RNP motifs of certainRRMs engage in protein–protein rather than protein–RNA recognition (reviewed in [37,38]), including theY14-mago [39–41] and Upf3–Upf2 [42] complexes.Remarkably, the TIA-1 RNP1 and RNP2 motifs interactextensively with the a-helices of a symmetry-related mole-cule (Fig. 3A). The buried solvent-accessible surface areais large for a crystal packing interface (945 A2 comparedwith 570 A2 average crystal packing interface), but slightlyless than expected for an oligomeric protein complex(1200–2000 A2) [43]. Despite low primary sequence homol-ogy (27%), the symmetry-related TIA-1 contacts are quali-tatively similar to the Y14 interactions with the a-helices ofthe mago partner [39–41] (Fig. 3B). In contrast, the Upf3–Upf2 orientation [42] differs from that of the symmetry-related TIA-1 RRMs. Although the TIA-1–RRM2 domainis likely to bind RNA in a canonical manor given its estab-lished role in RNA binding [19,21,22], the extensive sym-metry-related interface mediated by the TIA-1 RNPmotifs illustrates the versatile potential of RNPmotifs to engage in protein–protein as well as protein–RNA interactions.

3.4 Comparison of putative RNA binding surfaces

The close structural matches of TIA-1–RRM2 with theN-terminal RRMs of PAB and SXL provide an opportu-nity to compare the residues involved in RNA interactions.Six prominent residues responsible for RNA interactions inboth the PAB and SXL complexes are also conserved in theTIA-1–RRM2 (Fig. 3C–D), despite slight differencesamong the RNA sequences preferred by these factors.Three hydrophobic residues of the RNP motifs (RNP2-Phe98; RNP1-Tyr138; RNP1-Phe140) mediate conservedbase- and sugar-stacking interactions of PAB and SXL,

as well as the majority of other canonical RRMs [38].Despite amino acid conservation, the remaining three resi-dues are involved in distinct types of RNA interactions inthe PAB compared with SXL complexes, due to differencesin the local conformations of the RNA ligands. Mostprominently, an adenosine in the PAB complex is rotated180� relative to the SXL-bound uridine counterpart. Alto-gether, the PAB residues corresponding to Arg125,Asp129, and Arg167 of TIA-1, respectively, engage in basestacking interactions, a specific hydrogen bond with anadenosine exocyclic amine, and a phosphate salt bridge[25], whereas the corresponding residues of SXL, respec-tively, hydrogen bond with a ribose 20 hydroxyl, lack directRNA interactions, or form a specific hydrogen bond with auracil base [31]. These differences underscore the difficultiesconfronting accurate prediction of RRM–RNA interac-tions, despite an expanding number of RRM-nucleic acidstructures [44].

3.5 Locations of RNP residues implicated in nuclear import

Previously, the RRM2 RNP motifs of the TIA-1 homo-logue, TIAR, were mutated to investigate the importanceof RNA binding for nuclear import [23]. Given that theRRM2 regions of TIA-1 and TIAR share 93% sequenceidentity, the availability of the TIA-1–RRM2 structureprovides the opportunity to examine the locations of themutated residues (Fig. 4). The nuclear localization oftwo mutant proteins was investigated, each with acluster of mutations in either RNP1 (Gly137Asp/Tyr138-

818 A.O. Kumar et al. / Biochemical and Biophysical Research Communications 367 (2008) 813–819

Asp/Phe140Asp/Val141Glu, TIA-1 numbering), or RNP2(Phe98Asp/Gly100Pro/Leu102Asp). Although several ofthe residues are solvent-exposed and may interact withRNA, both Leu102 (RNP2) and Val141 (RNP1) are buriedin the hydrophobic protein interior (Fig. 4), where chargedaspartate and glutamate side chains introduced by the site-directed mutagenesis would be likely to interfere withdomain folding. Thus, the conclusion that TIAR RNAbinding ability is important for nuclear accumulation ispremature. Instead, the TIA-1–RRM2 structure presentedhere supports the conclusion that the folded RRM2domain is necessary for nuclear accumulation, as shownby deletion analysis of both TIA-1 and TIAR [23].

Acknowledgments

We thank J. Valcarcel for graciously sharing plasmids,and E.C. Reilly for protein purification. M.C.S. was sup-ported in part by a training grant from the National Can-cer Institute (CA009110). The Kimmel Foundation andNational Institutes of Health Grant R01-GM070503 toC.L.K. supported this work.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.bbrc.2008.01.027.

References

[1] J.M. Izquierdo, N. Majos, S. Bonnal, C. Martinez, R. Castelo, R.Guigo, D. Bilbao, J. Valcarcel, Regulation of Fas alternative splicingby antagonistic effects of TIA-1 and PTB on exon definition, Mol.Cell 19 (2005) 475–484.

[2] P. Forch, O. Puig, N. Kedersha, C. Martinez, S. Granneman, B.Seraphin, P. Anderson, J. Valcarcel, The apoptosis-promoting factorTIA-1 is a regulator of alternative pre-mRNA splicing, Mol. Cell 6(2000) 1089–1098.

[3] F. Del Gatto-Konczak, C.F. Bourgeois, C. Le Guiner, L. Kister,M.C. Gesnel, J. Stevenin, R. Breathnach, The RNA-binding proteinTIA-1 is a novel mammalian splicing regulator acting through intronsequences adjacent to a 50 splice site, Mol. Cell. Biol. 20 (2000)6287–6299.

[4] S. Shukla, F. Del Gatto-Konczak, R. Breathnach, S.A. Fisher,Competition of PTB with TIA proteins for binding to a U-rich cis-element determines tissue-specific splicing of the myosin phosphatasetargeting subunit 1, RNA 11 (2005) 1725–1736.

[5] E. Zuccato, E. Buratti, C. Stuani, F.E. Baralle, F. Pagani, An intronicpolypyrimidine-rich element downstream of the donor site modulatescystic fibrosis transmembrane conductance regulator exon 9 alterna-tive splicing, J. Biol. Chem. 279 (2004) 16980–16988.

[6] A. McAlinden, L. Liang, Y. Mukudai, T. Imamura, L.J. Sandell,Nuclear protein TIA-1 regulates COL2A1 alternative splicing andinteracts with precursor mRNA and genomic DNA, J. Biol. Chem.282 (2007) 24444–24454.

[7] I. Lopez de Silanes, S. Galban, J.L. Martindale, X. Yang, K. Mazan-Mamczarz, F.E. Indig, G. Falco, M. Zhan, M. Gorospe, Identifica-tion and functional outcome of mRNAs associated withRNA-binding protein TIA-1, Mol. Cell. Biol. 25 (2005) 9520–9521.

[8] M. Piecyk, S. Wax, A.R. Beck, N. Kedersha, M. Gupta, B. Maritim,S. Chen, C. Gueydan, V. Kruys, M. Streuli, P. Anderson, TIA-1 is a

translational silencer that selectively regulates the expression of TNF-alpha, EMBO J. 19 (2000) 4154–4163.

[9] D.A. Dixon, G.C. Balch, N. Kedersha, P. Anderson, G.A. Zimmer-man, R.D. Beauchamp, S.M. Prescott, Regulation of cyclooxygenase-2 expression by the translational silencer TIA-1, J. Exp. Med. 198(2003) 475–481.

[10] S. Yamasaki, G. Stoecklin, N. Kedersha, M. Simarro, P. Anderson,T-cell intracellular antigen-1 (TIA-1)-induced translational silencingpromotes the decay of selected mRNAs, J. Biol. Chem. 282 (2007)30070–30077.

[11] N. Kedersha, P. Anderson, Stress granules: sites of mRNA triage thatregulate mRNA stability and translatability, Biochem. Soc. Trans. 30(2002) 963–969.

[12] C. Le Guiner, M.C. Gesnel, R. Breathnach, TIA-1 or TIAR isrequired for DT40 cell viability, J. Biol. Chem. 278 (2003) 10465–10467.

[13] A.R. Beck, I.J. Miller, P. Anderson, M. Streuli, RNA-binding proteinTIAR is essential for primordial germ cell development, Proc. Natl.Acad. Sci. USA 95 (1998) 2331–2336.

[14] A.R. Beck, Q.G. Medley, S. O’Brien, P. Anderson, M. Streuli,Structure, tissue distribution and genomic organization of the murineRRM-type RNA binding proteins TIA-1 and TIAR, Nucleic AcidsRes. 24 (1996) 3825–3829.

[15] A. Kawakami, Q. Tian, M. Streuli, M. Poe, S. Edelhoff, C.M.Disteche, P. Anderson, Intron-exon organization and chromosomallocalization of the human TIA-1 gene, J. Immunol. 152 (1994) 4937–4945.

[16] A. Kawakami, Q. Tian, X. Duan, M. Streuli, S.F. Schlossman, P.Anderson, Identification and functional characterization of a TIA-1-related nucleolysin, Proc. Natl. Acad. Sci. USA 89 (1992) 8681–8685.

[17] J.M. Izquierdo, J. Valcarcel, Two isoforms of the T-cell intracellularantigen 1 (TIA-1) splicing factor display distinct splicing regulationactivities. Control of TIA-1 isoform ratio by TIA-1-related protein, J.Biol. Chem. 282 (2007) 19410–19417.

[18] N. Gilks, N. Kedersha, M. Ayodele, L. Shen, G. Stoecklin, L.M.Dember, P. Anderson, Stress granule assembly is mediated by prion-like aggregation of TIA-1, Mol. Biol. Cell 15 (2004) 5383–5398.

[19] P. Forch, O. Puig, C. Martinez, B. Seraphin, J. Valcarcel, The splicingregulator TIA-1 interacts with U1-C to promote U1 snRNP recruit-ment to 50 splice site, EMBO J. 21 (2002) 6882–6892.

[20] E.A. Suswam, Y.Y. Li, H. Mahtani, P.H. King, Novel DNA-bindingproperties of the RNA-binding protein TIAR, Nucleic Acids Res. 33(2005) 4507–4518.

[21] L.M. Dember, N.D. Kim, K.Q. Liu, P. Anderson, Individual RNArecognition motifs of TIA-1 and TIAR have different RNA bindingspecificities, J. Biol. Chem. 271 (1996) 2783–2788.

[22] W. Li, Y. Li, N. Kedersha, P. Anderson, M. Emara, K.M. Swiderek,G.T. Moreno, M.A. Brinton, Cell proteins TIA-1 and TIAR interactwith the 30 stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication, J. Virol. 76 (2002) 11989–12000.

[23] T. Zhang, N. Delestienne, G. Huez, V. Kruys, C. Gueydan,Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins, J.Cell. Sci. 118 (2005) 5453–5463.

[24] A. Sali, T.L. Blundell, Comparative protein modeling by satisfactionof spatial restraints, J. Mol. Biol. 234 (1993) 779–815.

[25] R.C. Deo, J.B. Bonanno, N. Sonenberg, S.K. Burley, Recognition ofpolyadenylate RNA by the poly(A)-binding protein, Cell 98 (1999)835–845.

[26] L.C. Storoni, A.J. McCoy, R.J. Read, Likelihood-enhanced fastrotation functions, Acta Crystallogr. D60 (2004) 432–438.

[27] A.J. McCoy, R.W. Grosse-Kunstleve, L.C. Storoni, R.J. Read,Likelihood-enhanced fast translation functions, Acta Crystallogr.D61 (2005) 458–464.

[28] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros,R.W. Grosse-Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S.

A.O. Kumar et al. / Biochemical and Biophysical Research Communications 367 (2008) 813–819 819

Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren,Crystallography & NMR system: a new software suite formacromolecular structure determination, Acta Crystallogr. D54(1998) 905–921.

[29] C.C. Project, and N. 4, The CCP4 suite: Programs for proteincrystallography, Acta Crystallogr. D50 (1994) 760–63.

[30] P. Emsley, K. Cowtan, Coot: model-building tools for moleculargraphics, Acta Crystallogr. D60 (2004) 2126–2132.

[31] N. Handa, O. Nureki, K. Kurimoto, I. Kim, H. Sakamoto, Y.Shimura, Y. Muto, S. Yokoyama, Structural basis for recognition ofthe tra mRNA precursor by the sex-lethal protein, Nature 398 (1999)579–585.

[32] Y. Shamoo, U. Krueger, L.M. Rice, K.R. Williams, T.A. Steitz,Crystal structure of the two RNA binding domains of human hnRNPA1 at 1.75 A resolution, Nat. Struct. Biol. 4 (1997) 215–222.

[33] C. Mazza, M. Ohno, A. Segref, I.W. Mattaj, S. Cusack, Crystalstructure of the human nuclear cap binding complex, Mol. Cell 8(2001) 383–396.

[34] E. Bae, N.J. Reiter, C.A. Bingman, S.S. Kwan, D. Lee, G.N. PhillipsJr., S.E. Butcher, D.A. Brow, Structure and interactions of the firstthree RNA recognition motifs of splicing factor prp24, J. Mol. Biol.367 (2007) 1447–1458.

[35] H. Ohtaki, Ionic solvation in aqueous and nonaqueous solutions,Monatshefte fur Chemie 132 (2001) 1237–1268.

[36] E.A. Sickmier, K.E. Frato, C.L. Kielkopf, Crystallization andpreliminary X-ray analysis of U2AF65 variant in complex with apolypyrimidine tract analogue by use of protein engineering, ActaCrystallogr. F62 (2006) 457–459.

[37] C.L. Kielkopf, S. Lucke, M.R. Green, U2AF homology motifs:protein recognition in the RRM world, Genes Dev. 18 (2004) 1513–1526.

[38] C. Maris, C. Dominguez, F.H. Allain, The RNA recognition motif, aplastic RNA-binding platform to regulate post-transcriptional geneexpression, FEBS J. 272 (2005) 2118–2131.

[39] S. Fribourg, D. Gatfield, E. Izaurralde, E. Conti, A novel mode ofRBD-protein recognition in the Y14-Mago complex, Nat. Struct.Biol. 10 (2003) 433–439.

[40] C.K. Lau, M.D. Diem, G. Dreyfuss, G.D. Van Duyne, Structure ofthe Y14-Magoh core of the exon junction complex, Curr. Biol. 13(2003) 933–941.

[41] H. Shi, R.M. Xu, Crystal structure of the Drosophila Mago nashi-Y14 complex, Genes Dev. 17 (2003) 971–976.

[42] J. Kadlec, E. Izaurralde, S. Cusack, The structural basis for theinteraction between nonsense-mediated mRNA decay factors UPF2and UPF3, Nat. Struct. Mol. Biol. 11 (2004) 330–337.

[43] J. Janin, F. Rodier, P. Chakrabarti, R.P. Bahadur, Macromolecularrecognition in the Protein Data Bank, Acta Crystallogr. D Biol.Crystallogr. 63 (2007) 1–8.

[44] S.D. Auweter, F.C. Oberstrass, F.H. Allain, Sequence-specific bind-ing of single-stranded RNA: is there a code for recognition? NucleicAcids Res. 34 (2006) 4943–4959.

[45] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton,PROCHECK: a program to check the stereochemical quality ofprotein structures, J. Appl. Crystallogr. 26 (1993) 283–291.

[46] L. Holm, C. Sander, Protein structure comparison by alignment ofdistance matrices, J. Mol. Biol. 233 (1993) 123–138.