Structure of the Y14-Magoh Core of the Exon Junction Complex

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provided by Elsevier - Publisher Connector Current Biology, Vol. 13, 933–941, May 27, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00328-2 Structure of the Y14-Magoh Core of the Exon Junction Complex

Chi-Kong Lau, Michael D. Diem, Gideon Dreyfuss, process also alters the protein composition of the mRNP and Gregory D. Van Duyne* complex in an important way. In conjunction with each Howard Hughes Medical Institute and splicing event, a multisubunit complex of proteins is Department of Biochemistry and Biophysics assembled onto the mRNP particle near the location of University of Pennsylvania School of Medicine the exon-exon splice junction. This protein complex, Philadelphia, Pennsylvania 19104-6059 termed the exon-exon junction complex (EJC), binds ca. 24 bases upstream of the junction and serves to mark the locations of splicing activity and thus the prior loca- Summary tion of introns in the mature mRNA [7–9]. The EJC-mRNA interaction is strictly dependent upon splicing and has Background: Splicing of pre-mRNA in eukaryotes im- positional rather than sequence-based specificity. prints the resulting mRNA with a specific multiprotein The composition of the EJC changes during the complex, the exon-exon junction complex (EJC), at the postsplicing maturation of the mRNA. The core compo- sites of intron removal. The proteins of the EJC, Y14, nents of the complex as it first forms on the 5Ј exon Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical [10] during splicing include Aly/REF [11, 12] and the roles in postsplicing processing, including nuclear ex- cytoplasmic shuttling proteins Y14 [7] and Magoh [10, port and cytoplasmic localization of the mRNA, and the 13, 14]. Other identified components of the nuclear EJC nonsense-mediated mRNA decay (NMD) surveillance include RNPS1, Srm160, and Upf3 [8, 9, 15, 16]. process. Y14 and Magoh are of particular interest be- Y14 is a 20 kDa nuclear shuttling protein with a central cause they remain associated with the mRNA in the RNA binding domain (RBD; also referred to as an RRM same position after its export to the cytoplasm and re- or RNP domain; [17]) that was first identified in a two- quire translation of the mRNA for removal. This tenacious, hybrid screen by using the nuclear import protein RanBP persistent, splicing-dependent, yet RNA sequence-inde- as bait [7]. Y14 and Magoh are associated preferentially pendent, association suggests an important signaling with mRNAs produced by splicing, but they are not effi- function and must require distinct structural features for ciently associated with pre-mRNAs, introns, or mRNAs these proteins. produced by genes lacking introns [7, 13, 14]. Y14 and Results: We describe the high-resolution structure and Magoh are found tightly associated with one another biochemical properties of the highly conserved human outside of the EJC and are imported into the nucleus Y14 and Magoh proteins. Magoh has an unusual struc- as a complex [18]. After EJC formation and nuclear ex- ture comprised of an extremely flat, six-stranded anti- port, Y14 and Magoh remain anchored to their initial parallel ␤ sheet packed against two helices. Surpris- mRNA binding sites until their eventual removal by active ingly, Magoh binds with high affinity to the RNP motif translation of the mRNA in the cytoplasm [19]. RNA binding domain (RBD) of Y14 and completely Magoh is the human homolog of the Drosophila mago masks its RNA binding surface. nashi gene product [20, 21]. A single point mutant in Conclusions: The structure and properties of the Y14- the mago nashi locus gives rise to a “grandchildless” Magoh complex suggest how the pre-mRNA splicing phenotype in flies due to a defect in the correct cyto- machinery might control the formation of a stable EJC- plasmic localization of oscar mRNA [20, 22, 23]. In this mRNA complex at splice junctions. mutant, the posterior lobe of the fly embryo is not prop- erly developed, and viable egg sacs are not formed Introduction in female offspring. Magoh is a small (18 kDa), highly conserved protein with no recognizable relationship to The expression of protein-coding genes in eukaryotes known sequences. The Drosophila ortholog (Tsunagi) is a complex, multistep process involving transcription has also been characterized, and, like Mago nashi, it is of pre-mRNA by RNA polymerase II, removal of introns essential for proper localization of oscar mRNA [24, 25]. by pre-mRNA splicing, 5Ј-end capping, 3Ј-end cleavage Both Y14 and Magoh are expressed in all tissues [21], and polyadenylation, nuclear export, cytoplasmic local- and Magoh is an essential gene in Drosophila [20] and ization, and finally translation into protein by ribosomes. C. elegans [26]. At all stages of its life, an mRNA transcript exists as a The EJC provides an important link between nuclear complex with a variety of RNA binding proteins, and the processing of pre-mRNA and at least three distinct composition of this mRNP particle is highly dynamic downstream activities: nuclear export, nonsense-medi- (reviewed in [1–3]). Several of the mRNA-processing ated mRNA decay (NMD), and cytoplasmic localization steps are monitored by surveillance mechanisms that (reviewed in [1, 27]). For some genes, it has been demon- act to prevent inappropriate or premature gene expres- strated that splicing leads to an increase in nuclear ex- sion (reviewed in [4–6]). port efficiency relative to the same gene lacking introns Recent studies have demonstrated that, in addition [28]. The nuclear export factor TAP has been shown to to removing introns from pre-mRNA, the RNA splicing associate with nuclear EJCs [12, 15, 29, 30] and to interact with isolated Y14 and Magoh in vitro [13]; these *Correspondence: [email protected] findings indicate that the EJC may provide explicit ex- Current Biology 934

port factor docking sites that facilitate the transport of spliced mRNA to the cytoplasm. A central role for the EJC in nonsense-mediated mRNA decay is even more clearly established. In this process, mRNAs containing premature stop codons are selected for degradation in order to avoid repeated translation into harmful protein truncations. The identification of a premature termination codon was shown to be related to the presence of downstream exon-exon junctions in earlier studies (reviewed in [4, 31–33]). In a more recent model for NMD in the context of EJC- imprinted mRNA [1], the ribosome, or associated factors, removes EJCs as it translates through exon-exon boundaries. If an in-frame stop codon is encountered Figure 1. Binding Titration Curves for Y14-Magoh and Y14t-Magoh on an mRNA containing a downstream EJC, then that Binding was monitored by fluorescence resonance energy transfer mRNA is targeted for destruction by surveillance factors between donor-labeled Y14 (or Y14t) and acceptor-labeled Magoh. ϭ Ϯ activated as a result of termination. Since stop codons Dissociation constants are Kd 1.8 0.2 nM for Y14t-Magoh and K ϭ 0.7 Ϯ 0.1 nM for Y14-Magoh. generally occur in the final exon of protein-coding genes, d the presence of EJCs downstream of a stop codon provides the signal for premature termination. The Upf3 and identification of the resistant fragments showed that RNPS1 proteins are each implicated in NMD activity and Magoh is stable to gentle proteolysis but that Y14 is have been identified as components of the EJC [6, 16]. rapidly converted to a 12 kDa fragment in the presence The molecular details of the dynamic protein-protein of Magoh. Edman sequencing of the N terminus of trun- and protein-mRNA interactions present during the life- cated Y14 confirmed that the polypeptide comprised time of the EJC are the subject of ongoing investigations. amino acids Met50–Lys168. The resulting complex of A central role for the Y14 and Magoh proteins in this truncated Y14, referred to here as Y14t, and full-length complex, however, is now clear. In order to establish a Magoh was further purified with anion-exchange and structural framework for understanding the function of size-exclusion chromatography. the EJC, we have begun an investigation into the structural biochemistry of this system, starting with the Y14- Magoh complex. Y14 and Magoh Form an Extremely Here, we report the structure, binding affinity, and Stable Heterodimer oligomeric state of the human Y14-Magoh complex. The To determine the energetics of the Y14-Magoh interac- Magoh protein has a novel ␣/␤ structure containing a tion, we analyzed the binding of Magoh to Y14 and flat, six-stranded ␤ sheet packed against two ␣ helices. Y14t in solution by using time-resolved fluorescence The helical face of Magoh forms an extensive, primarily resonance energy transfer. Fluorescent donor mole- hydrophobic interface with the RBD of Y14 and com- cules were attached to a GST fusion of Y14 (or Y14t) via pletely occludes the canonical mRNA binding surface. labeled anti-GST antibodies, and fluorescent acceptors The strongly conserved ␤ sheet surface of Magoh, com- were attached to hexahistidine-tagged Magoh via la- bined with an equally well-conserved concave pocket beled anti-His antibodies (see the Experimental Proce- formed as a consequence of the extended sheet, pro- dures). The results of these binding experiments are vides interaction surfaces that may be used to bind other shown in Figure 1. Both full-length and truncated Y14 EJC components, mRNA, or adaptor proteins used in bind with nanomolar affinity to Magoh. As discussed mRNA transport or specific surveillance activities. The later, the increased binding affinity for full-length Y14 structure of the Y14-Magoh complex suggests that Ma- compared to Y14t is most likely due to the presence of goh inhibits Y14’s ability to interact with mRNA in the an additional Magoh binding surface in the N-terminal absence of splicing and that components of the splicing 50 amino acids of Y14 that are removed by trypsin diges- machinery may open the Y14-Magoh complex and re- tion in Y14t. veal the surfaces required for high-affinity interaction To investigate the oligomeric state of the Y14-Magoh with the appropriately positioned segment of mRNA in and Y14t-Magoh complexes, both were analyzed by the 5Ј exon. sedimentation equilibrium analytical ultracentrifugation. At concentrations of 10–20 ␮M, both complexes are stable heterodimers, and no dissociation of subunits or Results and Discussion oligomerization of the heterodimers is observed (data not shown). Formation of higher-order oligomers of the A Protease-Resistant Y14-Magoh Subcomplex Y14-Magoh heterodimer in the context of the mRNA The Y14-Magoh complex can be overexpressed in solu- bound EJC, however, cannot be ruled out. ble form in bacteria by coexpression of the two proteins [14]. Crystallization trials based on the full-length Y14- Structure Determination Magoh complex failed to produce diffraction-quality Diffraction-quality crystals of the Y14t-Magoh complex crystals. Therefore, limited proteolysis was used to iden- were first identified by using material produced by pre- tify a stable subcomplex. Trypsin digestion of the Y14- parative proteolysis, then later by crystallization of the Magoh complex and MALDI-TOF mass spectrometric corresponding recombinant Y14t-Magoh complex pro- Structure of Y14-Magoh Complex 935

Table 1. Crystallographic Data Diffraction Dataa Native Gd ␭1Gd␭2Gd␭3 Resolution (A˚ ) 54–2.0 50–3.0 50–3.0 50–3.0 Completeness (%) 97 (83) 94 (97) 95 (96) 96 (96) Redundancyb 6.5 (4.6) 2.8 (2.4) 2.8 (2.4) 2.8 (2.4) c Rsym (%) 7.0 (22.5) 7.8 (26.4) 7.8 (27.1) 8.7 (33.4) Multiwavelength Phasinga

Gd ␭1Gd␭2Gd␭3 Isomorphous Phasing Powerd 2.26 (1.36) 1.33 (0.56) – Anomalous Phasing Powerd 2.82 (1.25) 3.33 (1.48) 2.01 (0.94) Figure of Merite 0.58 (0.34) Refinement

Rworking (%) 21.9 (27.2)

Rfree (%) 26.9 (32.3) Average B (A˚ 2) 21.8 R.m.s. bonds (A˚ ) 0.017 R.m.s. angles (Њ) 1.65 a Values in parentheses are for the highest resolution shell. Wavelengths are ␭1 ϭ 1.7114 A˚ (inflection), ␭2 ϭ 1.7113 A˚ (peak), and ␭3 ϭ 1.6755 A˚ (remote). b Redundancy ϭ the number of observations/the number of unique reflections. c Rsym ϭ⌺I|Ih ϪϽIϾh|/⌺Ih, where ϽIϾh is the average intensity over symmetry equivalents. d Phasing power ϭϽ|Fh(calc)|/phase-integrated lack of closureϾ. e Computed prior to solvent flattening.

duced by bacterial coexpression. The structure was exposing both surfaces of the sheet to solvent. A con- phased at 3 A˚ by using multiwavelength anomalous scat- cave pocket is formed between the ␤ sheet extension tering data from three bound Gd ions in a soaked Gd and helix ␣3 as a consequence (Figure 3). derivative and was then refined to 2.0 A˚ resolution by Two segments containing heptad repeats of hy- using native data. Crystallographic data and the results drophobic side chains [21] correspond to the ␣ helices of phasing and refinement are summarized in Table 1, and in Magoh. These helices are antiparallel and form an representative electron density is shown in Figure 2C. extensive hydrophobic interface with one another and There are two Y14t-Magoh heterodimers in the asym- with the interior surface of the flat ␤ sheet. There are metric unit of a P21 unit cell that interact with one another no large, extended loops between secondary structural via partial stacking of Magoh’s flat ␤ sheet surface. This elements in Magoh; in part, this explains the resistance A˚ 2 of solvent-accessible surface to trypsin proteolysis despite the presence of 21 Lys 750ف interface buries per heterodimer and is primarily composed of polar and and Arg residues out of a total of 146. The hydrophobic electrostatic interactions. Sedimentation equilibrium core of Magoh is rich in aromatic amino acids and has analysis of both full-length and truncated Y14-Magoh 18 total Phe, Tyr, and Trp residues in the sequence. complexes indicates that this is a crystal-packing inter- A search for similar three-dimensional structures with action and is unlikely to be physiologically significant. the DALI server [34] indicates that the Magoh domain structure is unique. The closest match is ␤2-microglobu- Magoh Forms a Novel Structure Containing lin (HLA-B8; PDB code 1AGD), in which the rather flat a Flat ␤ Sheet surface of the MHC peptide binding groove matches The Magoh primary sequence is extremely well con- well with the central part of the Magoh ␤ sheet. As served from humans to S. pombe (Figure 2A), and se- expected from the high level of sequence identity among quence- or structure-based searching algorithms do not Magoh orthologs, conservation maps over the entire predict a likely three-dimensional structure. The struc- Magoh structure (Figure 3). The hydrophobic core is ture of Magoh in the Y14 bound form is shown in Figure completely conserved, as are most of the residues that 2B. A six-stranded antiparallel ␤ sheet packs against interact with Y14 (discussed below). What is most sur- two antiparallel ␣ helices in a highly unusual manner. prising, however, is that the solvent-exposed flat surface This ␤ sheet is atypical because it is almost entirely of the ␤ sheet is almost entirely conserved among known untwisted and the individual strands are largely un- Magoh sequences. This conservation may be related to curved. These features lead to an extremely flat, ex- the formation of specific sites of interaction with other tended ␤ sheet surface. Because of the lack of right- EJC proteins, other RNA-processing adaptor proteins, handed twist and the lack of curvature in the strands, or with the splice junction region of mRNA. The high the sheet does not wrap around the helical subdomain degree of conservation may also relate to the need to as is commonly seen in small ␣/␤ domains. Instead, conserve the Magoh domain structure, and the specific roughly two-thirds of the sheet packs against the heli- pattern of ␤ sheet surface residues that are present may ces, and the remaining third of the sheet is extended, play important structural roles. The ␤ sheet surface of Current Biology 936

Figure 2. Structure of Human Magoh (A) Alignment of a diverse subset of known Magoh sequences. The shaded regions indicate Ն80% sequence identity. Secondary structure assignments are shown above the alignments, and residues contacting Y14 (Ͻ 3.8 A˚ ) are marked with an asterisk. Hs, human; Dm, D. melanogaster; Ce, C. elegans; At, A. thaliana; Sp, S. pombe. (B) A ribbon diagram of Magoh as seen bound to Y14.

(C) ␴A-weighted 2Fo-Fc electron density for the refined structure at 2 A˚ resolution, contoured at 1.4 ␴. The orientation of the indicated ␤ strands is the same as that shown in (B). The molecular drawings in Figures 2–6 were prepared with PYMOL [46].

Magoh is highly charged, with numerous acidic and ba- 4). Y14 sequences are not as strongly conserved as is sic side chains, but the surface also contains the strictly seen in Magoh orthologs, but the level of sequence conserved aromatic amino acids Tyr6, Tyr10, and identity is still quite high within the RBD. The RNP1 Phe21. and RNP2 motifs characteristic of RBDs are strongly conserved among Y14 orthologs and match extremely The RNA Binding Domain of Y14 well with consensus sequences for these common RNA As predicted from its amino acid sequence [7], Y14 con- binding modules [17]. A superimposition of the Y14 tains an RBD [35] that includes residues 72–149 (Figure structure (as observed complexed with Magoh) on the

Figure 3. Sequence Conservation Mapped onto the Magoh Structure Light-blue residues correspond to the shaded regions in Figure 2A (Ն80% identity), and dark-blue residues are less conserved. (A) Same orientation as in Figures 2B and 2C. (B) View from the opposite face, showing the concave surface formed by the ␤ sheet extension. Structure of Y14-Magoh Complex 937

Figure 4. Structure of Human Y14 (A) Alignment of Y14 sequences from the same organisms as shown in Figure 2. Shaded regions indicate 100% identity. Secondary structure assignments are shown above the alignments, and those residues that contact Magoh (Յ3.8 A˚ ) are indicated below the alignments with a asterisk. Vertical arrows indicate the sites of trypsin cleavage. (B) A ribbon diagram of Y14t as seen bound to Magoh. Conserved RNP1 and RNP2 motifs are colored brown. (C) The second RBD (RBD2) from the Sxl-RNA crystal structure ([37]; PDB code 1B7F) with only three residues of the bound oligonucleotide shown.

U1A/RNA complex crystal structure [36] and the Sxl- illustrated in Figure 5. The conserved RNA binding sur- RNA structure (comparing RBD2) [37] indicates that the face of Y14 is entirely occluded by the helical face of A˚ 2 2400ف structures are quite similar; the best overlap is observed Magoh in the complex. This interface buries for the RNP1 and RNP2 motifs. The second RBD of Sxl of solvent-accessible surface, most of which is hy- is shown in Figure 4C for comparison. Although Y14 drophobic in character. The amino acids involved in residues 50–168 are present in the Y14t-Magoh complex the Y14-Magoh interaction (colored red in Figure 5) are crystals, only residues 64–165 are sufficiently well or- strongly conserved in both proteins (Figures 2, 3, and dered to be visualized in the electron density. In the 4), and, in Y14, they include those side chains expected N-terminal region of Y14 removed by proteolysis, sec- to interact with RNA by comparison to other character- ondary structure predictions suggest a strong propen- ized RBDs [35]. Shape complementarity analysis of the sity for residues 23–35 to form an ␣ helix. Y14-Magoh interface gives a coefficient of 0.70, which In addition to the well-conserved RNP1 and RNP2 is indicative of a highly specific interface [39]. motifs, the region preceding RNP1 is also strongly con- In addition to the RNP1 and RNP2 motifs and the served among Y14 sequences (Figure 4). This segment peripheral residues shown in Figure 5C (e.g., Arg108 corresponds to “loop 3” in the canonical RBD, which is and Arg109), the C-terminal residues of Y14’s RBD also known to play a role in RNA binding [35]. Curiously, this play a major role in formation of the interface with Ma- loop is much smaller in the nuclear export factor TAP’s goh. For example, Cys149 and Phe150 of Y14 are entirely RBD, where it is only two residues in length [5, 38]. buried in a hydrophobic pocket of Magoh that is formed between the N-terminal end of helix ␣1 and the C-ter- The Y14-Magoh Interface minal end of ␣3. These residues are likely to provide The Y14t-Magoh complex structure and the residues specificity in the Y14 RBD-Magoh interaction surface, involved in the interface between the two proteins are given that most RBDs would be expected to make simi- Current Biology 938

Figure 5. The Human Y14t-Magoh Complex (A) A ribbon and transparent surface drawing of the complex. The helical face of Magoh covers the RNA binding surface of Y14 (com- pare to Sxl-RNA in Figure 4C). (B and C) The Y14 and Magoh components of the complex have been separated, reveal- ing the surfaces that form the interface. Con- tact residues are colored red and are indicated in Figures 2A and 4A. Selected residues are indicated for reference.

lar interactions with Magoh via their conserved RNP1 formed between the ␤ sheet and ␣3 (Figure 6). Both of and RNP2 motifs. these surfaces are fully solvent exposed in the complex While the interaction surface formed between Y14t with Y14 and are therefore prime candidates for sites and Magoh in the crystal structure described here is of interaction with other components of the EJC, the substantial in terms of buried surface area and leads to 5Ј exon region of spliced mRNA, or effector proteins an extremely stable heterodimeric complex, it is clear involved in mRNA trafficking, nuclear export, and NMD. from the binding experiments shown in Figure 1 that Some of the exposed surface is likely to be used in additional interactions exist between full-length Y14 and binding additional sequence elements in the N terminus Magoh that are not revealed in the structure. The of Y14, as discussed above. N-terminal 73 amino acids of Y14 were alone sufficient The conserved, concave surface shown in Figure 6B to identify Magoh as a binding partner in a yeast two- has many of the properties expected of an RNA binding hybrid assay, and the same fragment is effective in GST- module. The center of the cleft is primarily hydrophobic pulldown assays with Magoh [13]. Some of this N-ter- but has an assortment of polar, hydrogen bonding side minal Y14-Magoh interface may include the residues chains (e.g., Asn36, Ser38, and Gln86) and aromatic side immediately preceding ␤1 of the RBD, which are both chains that could form base-stacking interactions strongly conserved in Y14 orthologs and contact Magoh (His13, Tyr17, Phe40, and Phe134). Around the periphery in the complex (e.g., Ser69–Glu71; Figure 3). It seems are the expected Lys/Arg residues (Lys16, Lys41, Lys103, unlikely, however, that this small segment would alone Lys130, and Lys144), which are generally provided by lead to a stable interaction. Along with the 2.5-fold en- loops in RBDs [35]. The surface has only a modest posi- hancement of binding gained with full-length Y14, this tive electrostatic potential (not shown), which is also the suggests that additional residues in the N-terminal part case with the RNA binding surface of Y14. of Y14 also interact with Magoh. Candidates for this Several mutations originally identified in the Drosoph- additional interaction region include residues 50–63, ila mago nashi locus were mapped to lesions in the which are present but disordered in the crystal structure, Mago coding region [20]. These include truncations and or a part of the 49 residues at the amino terminus that deletions that cause zygotic lethality as well as the were removed by proteolysis. The fact that the region mago1 mutant, which gives the “grandchildless” pheno- of Y14 N-terminal to the RBD is capable of independently type from which the name “mago nashi” was derived. forming a stable Magoh interaction carries interesting The mago1 mutation converts Gly19 (Gly18 in human implications for how the Y14-Magoh complex might be Magoh) to an arginine residue (Figure 6A). Although argi- activated for mRNA binding as a consequence of splic- nine could be accommodated in this position of the ing (discussed below). Magoh structure, it is clear that a modest change in backbone dihedral angles would be required to avoid Conserved Surfaces in the Y14-Magoh Complex steric clashes. The result would be a small remodeling Two remarkable features of the Magoh protein structure of the ␤ hairpin connecting strands 1 and 2 and a drastic are the conserved flat ␤ sheet surface and the large cleft change in the local surface properties of this region of Structure of Y14-Magoh Complex 939

Figure 6. The Concave Surface Formed between Magoh’s Flat ␤ Sheet Extension and Helix ␣3 (A) The Y14-Magoh complex, viewed from the opposite direction as shown in Figure 5A. The location of the Drosophila mago1 mutation (corresponding to Gly18→Arg in human Ma- goh) is indicated in the turn between ␤ strands 1 and 2. (B) A close-up of the concave surface; hydrophobic residues are colored gray, basic residues are colored blue, acidic residues are colored red, and polar residues are colored magenta. A selected subset of the conserved residues on the surface is indicated. Y14 is drawn as a yellow surface. the ␤ sheet surface. A second viable mago nashi mutant RNP2 binding surface of Y14 most likely becomes ac- (WE7) resulted in substituting Ile91 (Ile90 in human Ma- cessible. The RBD-Magoh interface would need to be goh) by Thr. This Ile side chain forms part of the hy- disrupted in order for this to happen, but the N-terminal drophobic core of Magoh, and Thr in this position would Y14-Magoh interaction could in principle remain intact be expected to be moderately destabilizing. Interest- if the two Magoh interaction surfaces of Y14 are con- ingly, the WE7 mutant is able to complement the grand- nected by a flexible linker. Y14-Magoh could therefore childless phenotype of mago1 [20]. be thought of as a type of clamp, with the segment preceding Y14’s RBD serving as the hinge. In this model, Implications for Splicing-Dependent RNA Binding the splicing machinery, or an associated factor, would Although several proteins have been identified as com- open the clamp and expose both the RNA binding sur- ponents of the EJC, the only one other than Y14 for face of Y14 and the helical RBD binding surface of which an interaction with Magoh has been demon- Magoh. strated is the nuclear export factor TAP. Magoh binds An interesting implication of such a model is that splic- avidly to TAP in vitro, but these experiments were per- ing-dependent opening of the Y14-Magoh clamp pro- formed in the absence of Y14 [13]. In light of the Y14t- vides not only a conserved RNA binding surface, but Magoh structure described here and the fact that TAP also exposes the RBD binding surface of Magoh, which has an RBD [5, 38], it is possible that Magoh also inter- could then be available to interact with other EJC pro- acts similarly with the RBD of TAP. If this is the case, teins or stabilize the interaction with the mRNA. It is the Y14-Magoh complex would be expected to have unlikely, however, that the RBD of Y14 is capable of much reduced affinity for TAP, relative to free Magoh. mediating an extremely stable mRNA interaction on its Preliminary experiments indicate that this is indeed the own. Most RBDs that bind RNA with little sequence case (unpublished data). Alternatively, Magoh may use specificity have only modest binding affinities and act its hydrophobic RBD binding surface to interact with in tandem with other domains to achieve a stable inter- other regions of TAP. action [35]. Indeed, Y14 alone binds RNA only weakly. The finding that Magoh binds with high affinity to the If one or more parts of the conserved, solvent-exposed RBD of Y14, combined with earlier results demonstrating surfaces of Magoh (such as that shown in Figure 6) are that the N terminus of Y14 also forms a stable interaction in fact an RNA binding surface, then Magoh could also with Magoh, provides a useful structural framework for interact directly with the mRNA, and the opened form considering models of highly stable, but splicing-depen- of the Y14-Magoh complex could use surfaces from dent, mRNA binding in the EJC. The Y14-Magoh com- both domains to create an avid and stable mRNA plex has very low affinity for RNA in vitro (not shown), complex. yet this complex is believed to form the stable core It is clear from the 1 nM binding Kd for the Y14 RBD- of the mRNA bound EJC. Perhaps the most intriguing Magoh interaction that a substantial amount of energy question raised by the Y14-Magoh structure is whether would need to be supplied in order to remodel the Y14- the Y14 RBD-Magoh interface remains intact in the RNA Magoh complex. An appealing aspect of the above bound EJC or is disrupted to expose the RNA binding model is that a single, high-affinity protein-protein sur- surface of Y14. It is possible, for example, that the do- face (Y14-Magoh) could be effectively replaced by both main organization of the Y14-Magoh complex within the a substantial protein-RNA surface and a new protein- EJC is similar to that described here, with no disruption protein surface. The presentation of a specific combina- of the RBD-Magoh interface and little participation of tion of macromolecular domains and surfaces at the Y14’s RBD in binding to the 5Ј exon near the exon-exon right time and place to compete with the Y14-Magoh junction. In this case, as yet unidentified proteins must interface may therefore be the basis for the splicing- play a critical role in forming and maintaining the stable dependent formation of the stable core of the EJC. Fi- EJC-RNA interaction. nally, although the Y14-Magoh clamp model we suggest Alternatively, and we believe more likely, Y14 provides has several attractive features, it is possible that the a major contribution to the mRNA binding interaction interaction of this protein heterodimer with the RNA in- (and perhaps Magoh as well). In this case, the RNP1/ volves entirely different interactions from those that can Current Biology 940

be currently surmised from the structure. For example, Y14 (or GST-Y14t) was preincubated with 2 nM anti-GST antibody the RNP1 and RNP2 surfaces of Y14 may not become labeled with Europium-W1024 (anti-GST-Eu donor), while 1 ␮M His- ␮ exposed, and, instead, other parts of Y14, such as the Magoh was preincubated with 1 M anti-6Xhis conjugated to Sure- light-Allophycocyanin (anti-His-APC acceptor) for 1 hr. His-Magoh/ amino- and carboxyl-terminal domains, as well as parts anti-His-APC was then serially diluted from 1 ␮M to 0.98 nM in a of Magoh may interact with the RNA. A structure of black 96-well Optiplate (Perkin Elmer) in binding buffer. An equal the protein-RNA complex identical to that produced by volume of either 2 nM GST-Y14/anti-GST-Eu or 2nM GST-Y14t/anti- splicing will be necessary to distinguish these possibil- GST-Eu was then added to the wells. Binding was allowed to reach ities. equilibrium, and the plate was read with an excitation wavelength of 340 nm and an emission wavelength of 665 nm. Binding titration Experimental Procedures experiments were performed five times for both Y14-Magoh and Y14t-Magoh, and the average values were used to fit binding curves Expression and Purification of Y14-Magoh by using IgorPro (WaveMetrics). Data were fit by using a simple 1:1 ϭ Ϯ Ϯ A glutathione-S-transferase (GST) fusion of Y14 was coexpressed binding model, with Kd 1.8 0.2 nM for Y14t-Magoh and 0.7 with native, full-length Magoh overnight at 15ЊC by using compatible 0.1 nM for Y14-Magoh. plasmids in strain BL21(DE3). GST-Y14 was expressed from a modi- fied version of pET21a (Novagen) in which the pBR322 origin of Crystallization, Data Collection, and Structure Determination Њ replication had been replaced by the p15A origin from pACYC184, Crystals of Y14t-Magoh were grown at 21 C from hanging drops at and the GST-Y14 fusion coding region was cloned into NdeI and a concentration of 15 mg/ml in the presence of 100 mM sodium XhoI sites. Magoh was expressed from pET28a after insertion of cacodylate (pH 6.2), 25% 2-methyl-2,4-pentanediol, 20 mM CaCl2. the native coding region into NcoI and XhoI sites. After purification Large single crystals suitable for diffraction studies were obtained of the complex on glutathione-agarose resin (Sigma), the GST tag by microseeding and were flash-frozen in liquid nitrogen prior to was removed by dialysis overnight in the presence of tobacco etch data collection at 100K. The space group is P21. Cell constants are ϭ ˚ ϭ ˚ ϭ ˚ ␤ϭ Њ virus protease. The Y14-Magoh complex was further purified by as follows: a 47.2 A,b 108.5 A,c 50.9 A, 90.24 with anion-exchange chromatography with a MonoQ column (Phar- two Y14t-Magoh heterodimers per asymmetric unit. A nonisomor- macia) and then size-exclusion chromatography with a Bio-Rad phous Gd derivative was prepared by soaking in the presence of 1 SEC-250 column. The SEC elution time corresponds to a molecular mM gadolinium acetate for 2 hr. weight of 38 kDa relative to calibration with protein standards. The Multiwavelength anomalous scattering data were measured at Y14-Magoh complex was concentrated to Ն20 mg/ml in 10 mM Tris- the L-III absorption edge at NSLS beamline X4A by using a Rigaku HCl (pH 7.4), 250 mM NaCl by using Centricon YM-10 concentrators R-axis IV detector. High-resolution parent data were measured at (Millipore). Glutathione-S-transeferase (GST) fusions of Y14 and NSLS beamline X25 by using an ADSC Quantum IV CCD detector. Y14t and a 6Xhis fusion of Magoh were expressed overnight at 17ЊC Diffraction images were processed, and data were scaled by using by using a T7-polymerase system in E. coli strain BL21(DE3). GST- the HKL suite [41]. Three Gd sites identified from difference anoma- Y14 and GST-Y14t were purified by using glutathione-agarose resin lous Patterson maps were used for multiwavelength phasing in (Sigma). His-Magoh was purified by using Ni-NTA agarose (Qiagen). SHARP [42], followed by density modification with SOLOMON [43]. The electron density map was of high quality, and all four protein Limited Proteolysis chains were easily traced. Iterative rounds of model building with The Y14-Magoh complex at 4 mg/ml was incubated with increasing O [44] and refinement with REFMAC [45] converged rapidly to yield concentrations of trypsin in 100 mM Tris-HCl (pH 8), 0.1 mM DTT, the statistics given in Table 1. The final model includes Y14 residues 250 mM NaCl at 25ЊC for 30 min. Reactions were stopped, analyzed 64–155 and Magoh residues 2–145 for both complexes in the asym- by SDS-PAGE, and blotted on PVDF membrane. Bands correspond- metric unit and 98 water molecules. All residues have allowed values ␺ ing to large, stable fragments were analyzed by MADLI-TOF mass of (φ, ) backbone dihedral angles. spectrometry (Voyager DE-RP, PerSeptive Biosystems) after calibration of the instrument with cytochrome c and myoglobin stan- Acknowledgments dards. Assignment of fragment sequences from MALDI-TOF data was confirmed by Edman sequencing of blotted fragments (Keck We thank Dr. Naoyuki Kataoka, Dr. Mark Lemmon, and members of Biopolymer facility at Yale University). Preparative proteolysis of the Dreyfuss and Van Duyne laboratories for stimulating discussions Y14-Magoh was performed with 10 mg Y14-Magoh complex and 5 and Kaushik Ghosh for assistance with diffraction data collection. ␮g trypsin at room temperature for 30 min, followed by anion- G.D.V. also acknowledges Dr. Rui-Ming Xu for communication of exchange purification on a MonoQ column. Peak fractions were results prior to publication. Use of the National Synchrotron Light pooled, concentrated, and further purified on a SEC-250 column. Source beamlines X4A and X25 and assistance from beamline per- Pure Y14t-Magoh complex was concentrated to 10 mg/ml for crys- sonnel are gratefully acknowledged. The X25 beamline is supported tallization. by grants from the National Center for Research Resources of the National Institutes of Health and by grants from the Offices of Biolog- Analytical Ultracentrifugation ical and Environmental Research and of Basic Energy Sciences of Sedimentation equilibrium experiments were performed at 20ЊC with the Department of Energy. G.D. is an Investigator and G.D.V. is an an XL-A analytical ultracentrifuge (Beckman) and a Ti An60 rotor with Assistant Investigator of the Howard Hughes Medical Institute. six-channel epon charcoal-filled centerpieces and quartz windows. The Y14-Magoh complex (22 ␮M) was centrifuged at 12,000, 18,000, Received: March 20, 2003 and 28,000 rpm, and Y14t-Magoh complexes (13 ␮M) were centri- Revised: April 16, 2003 fuged at 12,000, 16,000, 20,000, and 24,000 rpm for 14 hr, when Accepted: April 22, 2003 equilibrium was confirmed. 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