[1] 16w Structural Insights into RNA Quality Control: The Ro Autoantigen Binds Misfolded RNAs via Its Central Cavity

[1] 197w The Ro 60 kDa autoantigen is a major target of the immune response in patients with systemic lupus erythematosus. In vertebrate cells, Ro binds misfolded small RNAs and likely functions in RNA quality control. In eukaryotes and bacteria, Ro also associates with small RNAs called Y RNAs. We present structures of unliganded Ro and Ro complexed with two RNAs at 1.95 and 2.2 Å resolution, respectively. Ro consists of a von Willebrand factor A domain and a doughnut-shaped domain composed of HEAT repeats. In the complex, a fragment of Y RNA binds on the outer surface of the HEAT-repeat ring, and singlestranded RNA binds in the toroid hole. Mutagenesis supports a binding site for misfolded RNAs that encompasses both sites, with a single-stranded end inserted into the toroid cavity. Our experiments suggest that one role of Y RNAs may be to regulate access of other RNAs to Ro. tein used in these studies. A.J.S. was supported by a Brown-Coxe fellowship and a fellowship from the Arthritis Foundation. This work was funded by grants from the G. Harold and Leila Y. Mathers Foundation, the Pew Charitable Trust (K.M.R.), and the NIH (R01-GM70521 to K.M.R. and R01-GM48410 to S.

[1] 128w Although many RNA molecules must fold into complex structures in order to function, even relatively small RNAs, such as tRNAs, 5S rRNAs, and the spliceosomal U snRNAs, have a propensity to misfold into nonfunctional conformations (Herschlag, 1995;Schroeder et al., 2004). Abnormal RNAs can also be generated in other ways, including transcriptional errors, posttranscriptional editing events, and incorrect processing. While multiple mechanisms by which defective or truncated mRNAs are recognized and targeted for degradation have been described (Maquat, 2004;Parker and Song, 2004), all these pathways require at least one round of translation, making them inaccessible to RNAs that are not translated. Far less is known of the quality-control mechanisms by which misfolded and otherwise abnormal noncoding RNAs are recognized or the fate (refolding versus degradation) of misfolded structural RNAs.

[2] 138w A protein that likely functions in a quality-control pathway for defective noncoding RNAs is the Ro 60 kDa protein (reviewed by Chen and Wolin, 2004). This RNA binding protein was first described as a major autoantigen in patients suffering from two systemic rheumatic diseases, systemic lupus erythematosus and Sjogren's syndrome. In patients with lupus, anti-Ro antibodies are highly associated with specific photosensitive skin lesions and with neonatal lupus, a syndrome *Correspondence: karin.reinisch@yale.edu in which mothers with anti-Ro antibodies give birth to infants with similar photosensitive skin lesions and a cardiac conduction defect, complete heart block (Patel and Werth, 2002;Buyon et al., 2004). Interestingly, mice lacking Ro develop a lupus-like syndrome consisting of glomerulonephritis, autoantibodies against chromatin and ribosomes, and sunlight sensitivity, suggesting that the normal function of Ro is important for prevention of autoimmunity (Xue et al., 2003).

[3] 70w In all cells that have been studied, Ro is both nuclear and cytoplasmic. In the cytoplasm, Ro associates with small RNAs of unknown function called Y RNAs. Orthologs of the human Ro/Y RNA complexes have been described in other mammals, the frog Xenopus laevis, the nematode Caenorhabditis elegans, and the eubacterium Deinococcus radiodurans (Hendrick et al., 1981;O'Brien et al., 1993;Farris et al., 1995;Van Horn et al., 1995;Chen et al., 2000).

[4] 148w Studies in vertebrate cell nuclei have revealed that Ro associates with misfolded small RNAs. In Xenopus oocytes, Ro binds a large population of variant pre-5S rRNAs that are inefficiently processed to mature 5S RNA and are eventually degraded (O'Brien and Wolin, 1994). The variant pre-5S rRNAs that Ro binds are longer at the 3# end than mature 5S rRNAs due to a failure of RNA polymerase III to terminate at the first transcription signal. These RNAs also contain point mutations that cause the RNAs to misfold into an alternative structure (O'Brien and Wolin, 1994;Shi et al., 1996). Recently, Ro was also found associated with misfolded variant U2 snRNAs in mouse embryonic stem cells (Chen et al., 2003). As Ro was the only protein described to interact with misfolded small RNAs in cells, Ro was proposed to function in quality control of defective noncoding RNAs (Chen et al., 2003).

[5] 132w In both mouse and bacterial cells, Ro is important for cell survival after ultraviolet (UV) irradiation. An ortholog of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival after UV irradiation (Chen et al., 2003). Consistent with a role in the handling of intracellular damage, both the Ro ortholog and the Y RNA are upregulated after irradiation. Furthermore, mouse embryonic stem cells lacking Ro display increased sensitivity to UV, but not ionizing, radiation. Following UV irradiation, both Ro and the Y RNA undergo alterations in their subcellular distribution, changing from largely cytoplasmic to mostly nuclear (Chen et al., 2003). Although the mechanism by which Ro facilitates cell survival after UV irradiation is unknown, it has been proposed that Ro targets UV-damaged RNAs for degradation (Chen et al., 2000;Chen et al., 2003).

[6] 171w Ro appears to interact with a variety of RNA structural elements. The binding site for Ro on Y RNAs is a highly conserved bulged helix (Wolin and Steitz, 1984;Pruijn et al., 1991;Green et al., 1998). As Ro binding is sequence specific and dependent on two bulges that widen the major groove, Ro was proposed to bind in the major groove of the conserved Y RNA helix (Green et al., 1998). In contrast, while Ro recognition of mis-folded pre-5S rRNAs requires the formation of an abnormal helix, neither the exact sequence of the helix nor the presence of bulges is important for binding (Shi et al., 1996). Moreover, Ro only binds misfolded pre-5S rRNAs that contain a 3# extension, which is not present in mature 5S RNA (O'Brien and Wolin, 1994). Despite the differences in the requirements for Ro binding to Y RNAs and misfolded pre-5S rRNAs, misfolded pre-5S rRNAs compete with Y RNA for binding, suggesting that the two RNAs bind to at least partly overlapping sites (Green et al., 1998).

[1] 212w The full-length Ro protein from Xenopus laevis was crystallized in both its unliganded and its RNA bound form. The structure of the unliganded protein was determined at 1.95 Å and has been refined to R = 21.3% and R free = 25.7%. The refined model includes residues 5-537, except for the disordered loops 136-143 and 336-340, and 489 water molecules. The structure of the Ro/RNA complex was determined at 2.2 Å. The crystallographic asymmetric unit contains two protein/RNA complexes. The protein model includes residues 5-537, except for those in the loop 336-340, which is disordered. In addition to protein, each complex consists of a fragment of Y RNA and also a single-stranded RNA oligonucleotide. We crystallized Ro in the presence of two strands of RNA that, when annealed, represent the minimal Ro binding site on Y RNA. It appears that the bound single-stranded RNA is one of the RNA strands that was inadvertently present in excess in the crystallization mix, and the modeled sequence is taken from the Y RNA. In one of the complexes, five of the residues in the single strand are ordered. In the second complex, there are seven ordered nucleotide residues. The two additional nucleotides are involved in crystal contacts. In addition to the protein/RNA complexes, the refined

[2] 171w Table 1. Refinement Statistics Ro/RNA Complex Resolution (Å) 20-2.2 R/R free (%) 20.2/23.8 Rmsd bond lengths (Å ´) 0.0064 Rmsd bond angles (°) 1.269 Rmsd improper angles (°) 0.745 Rmsd dihedral angles (°) 21.13 Rmsd on B factor: main chain/side chain 1.25/2.28 Ramachandran plot (2 complexes per asymmetric unit) Residues in most favored region 886 in additionally allowed region 78 in generously allowed region 4 in disallowed region 0 Number of water molecules 408 Unliganded Ro Resolution (Å) 20.0-1.95 R/R free (%) 21.3/25.7 Rmsd bond lengths (Å ´) 0.0081 Rmsd bond angles (°) 1.335 Rmsd improper angles (°) 0.854 Rmsd dihedral angles (°) 20.9 Rmsd on B factor: main chain/side chain 1.07/1.72 Ramachandran plot Residues in most favored region 440 in additionally allowed region 33 in generously allowed region 1 in disallowed region 0 Number of water molecules 489 model includes 408 water molecules, two magnesium ions, and two acetate ions from the crystallization buffer. The structure has been refined to R = 20.2% and R free = 23.8% (Table 1).

[3] 34w In both the unliganded Ro structure and the structure of the ribonucleoprotein complex, the Ro protein forms an elliptical toroid that measures 85 and 55 Å across and is 25-35 Å thick (Figure 1).

[4] 800w The elliptical central cavity measures w10-15 Å across. Ro consists of two domains. The domain that interacts with RNA is almost entirely helical and forms a closed loop (residues w30-360) that is clasped shut by the second domain (residues w5-30 and 360-537). The loop domain is reminiscent of HEAT-repeat proteins such as importin-β (Cingolani et al., 1999) or the PR65/A subunit of protein phosphatase 2A (Groves et al., 1999), though the arrangement of repeats is less regular in Ro, with distortion in the region encompassing helices H8 and H15. Except in this distorted region, the helices face the top and bottom surfaces of the toroid while the connecting loops line the central channel. Importin-β is similar to Ro in that it uses HEAT repeats to form a central cavity, in this case to enclose a domain of importin-α. In importin-β, however, these repeats are arranged into a continuous spiraling superhelix in which helices line the central cavity and interact in a modular fashion with residues of importin-α (Cingolani et al., 1999). The central channel of Ro and a narrow band of residues along the outer surface of the loop domain are basic (Figure 2B). In the structure of the Ro/RNA complex, a 20 nt fragment of Y RNA is bound on the outer, basic surface of the Ro loop domain, where it interacts with residues that are highly conserved in all Ro proteins (see below), while the ssRNA is bound in the central positively charged cavity (Figures 1A and 2). Residues in this cavity that interact with RNA are also well conserved. To our knowledge, Ro is the first protein shown to interact with nucleic acids using HEAT repeats. The pumilio homology domain interacts with single-stranded RNA ends using the related ARM repeats (Wang et al., 2002), but its RNA binding surface differs from both RNA binding surfaces in Ro. The second domain of Ro adopts a Rossmann fold similar to that of the vWFA domain found in the eponymous platelet-adhesion protein and also in the integrins (Whittaker and Hynes, 2002). Its core, a five-stranded parallel β sheet encompassing strands S5, S6, S7, S8, S10, S11, and S13, is sandwiched between two sets of parallel α helices, H23 and H24 and H21, H22, and H25 (Figure 1B). The plane of the sheet is normal to the long axis of Ro. Five highly conserved residues at the C-terminal edge of the β sheet form a divalent cation binding site, termed the MIDAS (metal-ion-dependent adhesion site) motif in integrins, where the motif was first described (see Shimaoka et al., 2002 for review). In the integrins, this motif (D-x-S-x-S…T…D) is a conformational switch that is critical both for transmitting changes that accompany ligand binding and for ligand binding itself. A glutamate or an aspartate from the ligand completes the MIDAS metal coordination. The metal coordination observed in Ro is similar to that in the ligand bound integrins (Figure 3). In the structure of the Ro/RNA complex crystallized in the presence of 100 mM magnesium acetate, a magnesium ion is directly coordinated by S378 and S380 of the MIDAS signature sequence D-x-S-x-S, T445, two water molecules, and an acetate ion (Figure 3B). The acetate may be a mimetic for a glutamate or an aspartate in an as-yetunknown interaction partner of Ro. Residues D376 and D469 of Ro coordinate one of the water molecules. The second water molecule in the Ro MIDAS motif is hydrogen bonded to Y47, a conserved residue near the N terminus of Ro. The metal coordination site is very similar in the unliganded Ro structure, except that, in the absence of magnesium acetate in the crystallization solutions, water molecules have been modeled into the Mg 2+ and the acetate sites. The thirty most N-terminal residues of Ro pack against the sides of the vWFA domain, and the N-terminal strands S1 and S2 form a small sheet with strand S12 in the vWFA fold. One surface of the vWFA domain, which includes the MIDAS motif, is highly acidic (Figure 2B). We note that other RNA binding proteins, whose structures have not yet been determined, may have a similar architecture to the Ro protein. Both the human TEP1 and its likely homolog Tetrahymena p80 share sequence homology with the HEAT-repeat domain of the Ro protein and also contain an adjacent vWFA domain (Anantharaman et al., 2002; Bateman and Kickhoefer, 2003). TEP1 is a component of large cytoplasmic ribonucleoprotein particles called vaults. Although the function of vaults is unknown, TEP1 binds small RNAs that are similar in size to Y RNAs (Kickhoefer et al., 2001). Although Ro residues critical for Y RNA binding are not conserved in TEP1, certain residues in the central cavity that interact with single-stranded RNA are conserved. Thus, TEP1/p80 may also fold into a toroid that interacts with single-stranded RNA.

[5] 192w In the Ro/RNA structure, the Y RNA fragment forms a frayed duplex in which the unpaired ends are part of a bulge in the complete w100 nt Y RNA (Figure 4). Helix H9 (182-187) in Ro is positioned as if to wedge apart the RNA stem. The unpaired nucleotides in the bulge, G9-A10 and U11-U12, are flipped away from the RNA stem and located to either side of this helix. The bases of A10 and G9 are stacked, as are those of U11 and U12. The side chain of L185 in helix H9 is intercalated between the bases of C8 and G9 and makes hydrophobic interactions with both. The conserved residue W177 is stacked with the G9 base. C8 in the stem is unpaired and interacts with N2 in the H187 side chain, which is at the C terminus of helix H9. C8 and the histidine imidazole stack over the adjacent base pair C7:G13 to form one end of the stem region. The H187 imidazole appears to be protonated since N1 is perfectly positioned to form a second hydrogen bond to U11 phosphate. C8 also interacts with the L185 carbonyl via N4.

[6] 209w Ro recognizes Y RNA both by specific interactions with the nucleotide bases and by shape complementarity. The major groove of the stem region adjacent to the bulge is widened to 18 Å, allowing for direct contacts with the bases. Almost all of the hydrogen bonding interactions are to nucleotide bases along one strand, specifically, to G4, G5, U6, C7, and C8, all of which are part of the helical stem region, and to G9 and A10 in the bulged region. The residues involved in hydrogen bonding to the nucleotide bases are highly conserved in all Ro proteins (Figures 2C, 2D, and 4). Critical residues include R146, which forms hydrogen bonds to both G4 and G5; R149, which forms hydrogen bonds to G5 and U6; and W144, which interacts with the C7 N4 via its carbonyl. The crucial role of H187 and its interaction with C8 is described above. D181 and K133 along with the main chain carbonyl oxygen of G176 interact with G9, a bulged residue, and K133 hydrogen bonds to the nucleotide base of A10. Interactions with the second strand are few, and only R146, which interacts with C15 by way of a water molecule, is conserved. Additionally, K200 forms a hydrogen bond with the base of U11.

[7] 84w Ro makes numerous contacts with the Y RNA backbone. Most of the residues in loops 142-149 and 176-187 are involved in hydrophobic or van der Waals contacts to the backbone or hydrogen bonds to the 2#OH groups. A number of residues (K108, Q109, K136, G142, K150, A147, and R184; see Figure 4) form hydrogen bonds to the phosphate groups of one RNA strand. Additionally, several basic residues near the protein/RNA interface (R113, K132, and R149) could contribute to electrostatic interactions with the charged phosphates.

[8] 69w Ro binding, we performed electrophoretic mobility shift assays (EMSA) using a mutant pre-5S that is bound by Ro (Shi et al., 1996). Removal of the 3# extension decreased the affinity of Ro for the RNA by more than tenfold (Figure 5A and data not shown). As the 3# extension is important for Ro binding to misfolded pre-5S rRNA, we propose that the 3# end binds in the inner hole.

[9] 308w To further explore the role of the central cavity in binding RNA, we mutated residues in the cavity that interact with single-stranded RNA in the Ro/RNA structure (K170A/R174A, R255S/R283S, and R120S) and tested their affinities for both Y RNA and misfolded pre-5S rRNA using EMSA. As would be expected if Y RNA does not bind in the central cavity, none of the mutations had a measurable effect on the affinity of Ro for Y RNA (Figure 5B and Table 2). In contrast, the double mutant K170A/R174A had an w7-fold decrease in affinity for misfolded pre-5S rRNA (Figure 5B and Table 2), confirming that residues in the cavity are important for Figure 5. Binding of Wild-Type and Mutant Ro Proteins to Y RNA and Misfolded Pre-5S rRNA (A) Effect of shortening the 3# end of pre-5S rRNA on Ro binding. 32 P-labeled misfolded X. laevis oocyte pre-5S rRNA (left) or pre-5S rRNA lacking 8 nt of the 3# trailer (right) was incubated without protein (lane 1) or with the indicated concentrations of Ro protein (lanes 2-9). The misfolded pre-5S rRNA contains several mutations that cause it to fold efficiently into the structure recognized by Ro (Shi et al., 1996). Protein/RNA complexes were separated from unbound RNA by native gel electrophoresis. (B) Binding of mutant Ro proteins to RNA. 32 P-labeled Xenopus Y3 RNA (left panels) or misfolded pre-5S rRNA (right panels) was incubated either without protein (lane 1) or with increasing amounts of wild-type Ro (top row) or the indicated mutant proteins (bottom four rows). Protein concentrations are given in nM above lanes 2-9. Protein/RNA complexes were separated from unbound RNA in native gels. binding this RNA. It is plausible that these residues interact with the phosphate backbone of the 5S rRNA single-stranded 3# end much as they interact with the single strand of RNA observed in the structure.

[10] 65w In contrast to K170A/R174A, several mutations had little effect on pre-5S rRNA binding. R255 and R283, which stack on either side of a cytosine base in the Ro/RNA complex and may contribute to electrostatic interactions with the single-stranded RNA phosphate backbone, do not appear to be important for binding misfolded pre-5S rRNA. Likewise, R120, which forms hydrogen bonds to a cytosine base in the Ro/RNA-

[11] 96w Table 2. Binding of Mutant Ro Proteins to RNA K D xY3 (nM) K D Pre-5S (nM) Wild-type 5.2 ± 2.5 25.0 ± 13.5 Residues that interact with Y RNA R146S R149S 21.9 ± 1.5 48.5 ± 14.4 H187S 153.2 ± 13.6 105.2 ± 20.2 R184A 30.8 ± 5.5 187.0 ± 40.2 K108A Q109A R184A 198.3 ± 10.3 135.3 ± 12.2 K136A 4.8 ± 2.3 22.3 ± 7.5 Residues in the central cavity K170A R174A 4.7 ± 2.6 179.5 ± 24.1 R255S R283S 3.4 ± 0.3 46.8 ± 0.2 R120S 5.6 ± 0.7 34.1 ± 4.8

[12] 86w complex structure, is not important for binding pre-5S rRNA. The absence of a mutation effect on pre-5S rRNA binding at these positions may suggest, as noted above, that the interactions with single-stranded RNA within the central cavity may vary for different RNA species. Furthermore, we show below that misfolded pre-5S rRNAs interact with an extensive surface on Ro not confined only to the central cavity. Thus, the effects of mutations that alter only a small part of the interaction surface may be expected to be subtle.

[13] 336w In addition to a free 3# end, Ro recognition of variant pre-5S rRNAs requires that the RNA misfold to form an alternative helix (Shi et al., 1996). As Ro binding protects this helix and adjacent helices from ribonuclease digestion (Shi et al., 1996), Ro likely also contacts helical elements in the misfolded RNA. We propose that these duplex portions of the misfolded 5S rRNA bind on the positively charged outer surface of the HEATrepeat domain, partly the same surface to which Y RNA binds. To explore the role of the Y RNA binding site in the binding of misfolded 5S rRNA, we mutated residues that interact with Y RNA in the Ro/RNA structure (H187S, R184A, K136S, R146S/R149S, and K108/Q109/ R184). We measured the affinities of the Ro mutants for both Y RNA and 5S rRNA by EMSA. Some of the mutations in the Y RNA binding site affected not only Y RNA binding, as expected, but also the binding of misfolded 5S rRNA (Figure 5B and Table 2). The H187S and K108A/Q109A/R184A mutants had the most significant effects on Y RNA binding, with w30-and w38-fold decreases in affinity, respectively. Their effects on misfolded 5S rRNA were more modest but still significant, with w4-to 5-fold decreases in affinity (Table 2). Residue R184 also appears to be important for both Y RNA and 5S rRNA binding, as the R184A mutant has an w6to 7-fold reduced binding affinity for both of these RNAs. Some residues that are important for Y RNA binding appear relatively unimportant for 5S rRNA binding. For example, mutating R146 and R149, residues involved in specific interactions with Y RNA bases, affects Y RNA binding but has little or no effect on 5S rRNA binding. Together these experiments suggest that although Y RNA and misfolded 5S rRNA may not interact with Ro in the same way, they bind to overlapping surfaces. These results are consistent with previous data revealing that misfolded pre-5S rRNA competes with Y RNAs for binding to purified Ro (Green et al., 1998).

[14] 50w Our mutagenesis results agree with a model in which duplex portions of the 5S rRNA bind to the positively charged outer surfaces of the Ro HEAT-repeat ring, including parts of the Y RNA binding site, while the single-stranded 5S rRNA terminus extends into the cavity and strengthens the binding interaction.

[15] 88w This model suggests that one role of Y RNAs may be to sterically block misfolded and other RNAs from binding to Ro. Possibly, then, Y RNA plays a regulatory role in small-RNA quality control in that its dissociation from Ro allows these misfolded RNAs to bind. The mechanism by which Y RNAs may dissociate from Ro is currently unclear. Conformational changes associated with single-stranded RNA binding, nuclear localization, and/or association with other proteins or cofactors could all result in a weakened affinity for Y RNA, allowing its dissociation.

[16] 199w The Ro protein was complexed with a series of RNA duplexes containing its recognition site. The RNA sequences varied in length and overhang. The best crystals were obtained with an RNA 20mer (see Figure 4D) using the hanging-drop vapor diffusion method. The reservoir solution contained 29% 2-methyl-2,4-pentanediol, 50 mM sodium acetate (pH 3.6), and 100 mM magnesium acetate. The drops were composed of 1 l of Ro/RNA complex at w10 mg/ml and 0.5 l of reservoir solution. Plate-shaped crystals grew to a maximum dimension of 300 × 100 × 100 m within a week. The crystals belong to space group P2 1 (a = 60.6, b = 153.4, c = 80.5 Å and β = 92.8°). The crystals diffract to 2.2 Å. In contrast to crystals of unliganded Ro, crystals of the Ro/RNA complex grew reproducibly from different batches of protein, and the selenomethionine-substituted protein crystallized readily. In preparation for heavy-atom soaking experiments, crystals were transferred to a solution containing 50 mM Tris-HCl (pH 7.0) and 1.25 mM TCEP in place of the sodium acetate and DTT in the crystallization solution. Crystals were loop mounted directly from this solution and flash frozen in liquid nitrogen prior to data collection.

[1] 115w We have proposed that the central basic cavity in Ro binds the 3# ends of misfolded RNAs while a positively charged, conserved region near the central cavity, which includes the Y RNA binding site, binds other portions of misfolded RNAs. One possibility is that Ro binding assists in destabilizing misfolded helices, facilitating either correct folding or degradation. Since vWFA domains involved in cell adhesion frequently mediate protein/protein interactions, we speculate that in Ro this domain serves as a platform for recruiting additional protein components of the quality-control pathway, such as helicases and/or ribonucleases. It is also conceivable that Ro assists these downstream compo-nents in their function, perhaps, given its toroidal shape, as a processivity factor.

[1] 104w Gel-purified RNA oligomers were purchased from Dharmacon. The oligomers were resuspended in buffer D (1.5 mM MgCl 2 , 125 mM NaCl, 20 mM Tris [pH 7.5]) and quantitated on the basis of absorbance at 260 nm. To form the Y RNA fragment, two strands of RNA were mixed in roughly stoichiometric quantities and annealed. The RNA was heated to 95°C for 2 min and then plunged into an ice bath. Ro was added to the RNA and incubated on ice for one hour. To determine the correct ratio of protein to RNA for crystallization, we performed gel shift assays (Batey et al., 2001).

[2] 150w Crystallization of the unliganded protein was difficult, as only some batches of protein crystallized, and the quality of the crystals varied. Rod-shaped crystals were grown at room temperature by hanging-drop vapor diffusion against a well containing 25% PEG 4000, 200 mM sodium formate, 30 mM NaH 2 PO 4 , and 100 mM Tris-HCl (pH 8.5). Crystals grew to a maximum dimension of 300 × 100 × 100 m within a week. They belong to space group P2 1 2 1 2 1 (a = 68.8, b = 91.7, and c = 93.9 Å). The best crystals diffracted to 1.8 Å. Crystals were mounted in a loop directly from the hanging drop and were flash frozen in liquid nitrogen prior to data collection. All data were collected at -160°C at beamline X25 at Brookhaven National Laboratory. Data were integrated, scaled, and merged using the program HKL2000 (Otwinowski and Minor, 1997).

[3] 15w Attempts to grow high-quality crystals of selenomethionine-substituted Ro were unsuccessful in the absence of RNA.

[4] 177w The structure of the Ro/RNA complex was determined by the multiple isomorphous replacement method with anomalous scattering. As a reference data set, we used data from a crystal in which the protein was 40% selenomethionine-substituted and in which four uridines of the RNA duplex were substituted with iodine at the C5 position (Figure 4D). Crystals grown with unsubstituted protein were not isomorphous with crystals grown from protein with R40% selenomethionine substitution. Nor were crystals grown with unsubstituted RNA isomorphous to those obtained with the iodinated RNA. We used the iodine anomalous signal from the reference crystal and data from three heavy-atom derivatives to determine the phases. The selenium derivative was prepared from the highly substituted selenomethionine protein. To prepare a lead and a uranyl derivative, we soaked a reference crystal in either 1 mM lead acetate or 1.5 mM uranyl for one day. Data sets were collected at beamlines X25 and X29 at Brookhaven National Laboratory (Table S1), and data were integrated, scaled, and merged using the program HKL2000 (Otwinowski and Minor, 1997) (Baldwin et al., 1998).

[5] 79w Prior to refinement using CNS (Brunger et al., 1998), 10% of all reflections were set aside to monitor R free . Noncrystallographic symmetry restraints were maintained in the initial stages of refinement, then released. Rounds of manual rebuilding were followed by cycles of torsion-angle dynamics, positional refinement, and individual B factor refinement. For the final model, R = 20.2% and R free = 23.8% over all data between 20.0 and 2.2 Å. Refinement statistics are listed in Table 1.

[6] 132w The structure of the unliganded Ro was determined by molecular replacement techniques as implemented in CNS (Brunger et al., 1998). As a search model, we used the structure of Ro in the refined ribonucleoprotein complex. The protein model was rebuilt with the help of omit maps. Since the crystallization solution did not contain divalent cations, and because other MIDAS-motif structures have been determined with water at the magnesium site (Baldwin et al., 1998), we have modeled water in place of a magnesium ion in the MIDAS-motif pocket. Rounds of rebuilding were followed by cycles of refinement as described for the Ro/RNA complex. For the final model, R = 21.3% and R free = 25.7% over all data between 20.0 and 1.95 Å. Data collection and refinement statistics are listed in Table 1.

[7] 18w Figures were prepared using the programs GRASP (Nicholls, 1993), Molscript (Kraulis, 1991), and O (Kleywegt and Jones, 1997).

[8] 14w Supplemental Data include one table and are available with this article online at http://www.cell.com/cgi/content/full/121/529/DC1/.

[1] 208w A single strand of RNA, apparently one of the strands used to form the Y RNA duplex, is bound in the basic central channel. In one complex in the asymmetric unit, the electron density is consistent with nucleotides U6-A10, while in the second complex, nucleotides G4 and G5 are also ordered by their participation in crystal contacts. The sequence encompassing U6-A10 is bound almost identically in both protein/RNA complexes in the asymmetric unit. The RNA is in an extended conformation that is stabilized by stacking interactions and van der Waals contacts with Ro (Figures 4E-4G): R255 and R283 stack on either side of C7, R318 makes van der Waals contact with the C8 base, M166 and R252 sandwich the G9 base, and L313 packs against the A10 base. Side chains from residues T123, K170, Y171, and R174 and the main chain nitrogen of L278 are involved in hydrogen bonds to the phosphate backbone, and several other basic residues (R255, R283, and R307) are close enough to the phosphate backbone for favorable electrostatic interactions. There are also hydrogenbond interactions to the C8 and G9 bases: R120 interacts with O2 and N3 of C8, while the main chain nitrogens of D275 and R252 interact with N1 and N2 of G9.

[2] 60w We do not yet know the degree of specificity with which Ro binds single-stranded RNA. The ordered binding in the crystals would indicate that some level of specificity may be possible. The predominance of backbone and stacking interactions argues for a low level of specificity, however. The plasticity of the binding site also suggests that many sequences might be accepted.

[3] 93w The central channel is wide enough to accommodate single-but not double-stranded RNA, and it is likely that short RNA segments simply thread into the channel. Such threading requires the single-stranded portion to have a free end. A loop, for example, could not bind. The use of a hole, or elongated cavity, to recognize ends has been previously observed in proteins that recognize peptide or nucleic-acid termini. The ringshaped lambda exonuclease, for example, can bind DNA only by sliding over the ends, biasing the enzyme to cleavage in terminal regions (Carter and Radding, 1971).

[4] 116w It is also possible, however, that the HEAT-repeat ring can open to allow RNA to enter the channel. For example, the vWFA domain could initially unclasp the Ro N terminus and then rebind it to encircle the RNA. Interestingly, the MIDAS motif in the vWFA domain is close to the interface with the N-terminal region of Ro, with one N-terminal residue, Y47, forming part of the secondary coordination sphere of the MIDAS magnesium ion. Conceivably, the MIDAS motif could play a role in conformational changes that accompany ring opening and closing. Transient opening of the loop might allow longer single-stranded regions to bind more rapidly and might allow even for the binding of large RNA loops.

[5] 87w The Ro protein was overexpressed in insect cells infected with recombinant baculovirus carrying the Xenopus laevis Ro cDNA (Green et al., 1998). To prepare mutants of Ro, the cDNA encoding X. laevis Ro (O' Brien et al., 1993) was inserted between the BamHI and HindIII sites in the pFastbac1 vector (Invitrogen). Mutations (Table 2) were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Plasmids were sequenced to confirm the presence of the expected mutation. Recombinant baculovirus was obtained using the Bac-to-Bac expression system (Invitrogen) following manufacturer instructions.

[6] 132w For both wild-type and mutant Ro, High Five cells were grown to a density of 1.5-2 × 10 6 cells/ml in Excel 405 medium (JRH), infected at 3-5 plaque-forming units/cell, and harvested 72 hr postinfection. Cells were resuspended in buffer A (100 mM NaCl, 25 mM Tris [pH 8.0], 50 mM DTT, 3 mM MgCl 2 , 1 mM PMSF) and lysed by sonication. Protein purification was as described (Green et al., 1998), except for high levels of DTT (w50 mM) present throughout purification. Briefly, the lysate was applied to a heparin column (Amersham) and eluted with a linear salt gradient in buffer B (20 mM Tris [pH 8.0], 10% glycerol, 50 mM DTT, 550 mM NaCl). Fractions containing Ro were pooled, concentrated, and loaded onto a Superdex200 gel filtration column (Amersham).

[7] 37w The protein was eluted in buffer C (100 mM NaCl, 20 mM Tris [pH 8.0], 10% glycerol, 50 mM DTT) and concentrated to 10-15 mg/ml. Yields from 1 L of insect cell culture varied between 5-10 mg.

[8] 151w After several attempts, a highly selenomethionine-substituted protein was expressed in insect cells. The High Five cells were infected as described above, but 8 hr postinfection, the Excel 405 medium was exchanged for a medium deficient in methionine and yeastolate (JRH). At about 20 hr postinfection, seleno-L-methionine (Sigma) was added to a final concentration of 50 mg/ml. Cells were harvested and protein purified as above. The yield from 1 L of insect cell culture was w3 mg. Several attempts to quantitate selenium substitution by electrospray mass spectrometry were unsuccessful. A protein that was only 40% seleno-substituted as determined by electrospray mass spectrometry was produced when Excel 405 deficient in methionine but not yeastolate was used as a depletion medium and the depletion period was 7 hr instead of 12. Selenomethionine-substituted Ro was purified like the native protein, except that 1.25 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used in place of DTT.

[9] 102w The clones containing Xenopus Y3 RNA (xY3 RNA) and misfolded pre-5S rRNA (pre-5S stem mt 1 + 2 RNA; Shi et al., 1996) under control of the T7 promoter have been described (Shi et al., 1996;Green et al., 1998). After linearization with DraI, xY3 RNA and pre-5S rRNA were synthesized in the presence of [α-32 P]rUTP (400 Ci/ mmol, Amersham) using T7 RNA polymerase as described (Shi et al., 1996). To generate misfolded pre-5S rRNA lacking the last 8 nucleotides, a DNA template for T7 transcription was synthesized from overlapping DNA oligonucleotides. All RNAs were gel purified and quantitated by scintillation counting.

[10] 207w For gel shift assays, purified RNAs were resuspended in binding buffer (20 mM Tris-HCl [pH 7.5], 125 mM NaCl, 1 mM EDTA, 1.5 mM MgCl 2 , 2 mM DTT, 10% glycerol), refolded by heating to 90°C for 2 min, and frozen on dry ice. After thawing slowly on ice, the RNAs were diluted to 1.5 fmol/l. Binding reactions contained 1.5 fmol xY3 RNA or misfolded pre-5S rRNA and 24 to 6144 fmol of native Ro protein or a mutant Ro protein, 0.3 g/L E. coli tRNA, and 0.01% NP-40 in binding buffer. The total reaction volume was 10 l. Reactions were incubated at room temperature for 30 min and then at 4°C for 30 min and loaded onto 5% polyacrylamide (38:2 acrylamide:bisacrylamide)/5% glycerol gels running at 2 V/cm (prerun at 16 V/cm for 10 min at 4°C). Gels were run at 4°C for 10 min at 2 V/cm, followed by 16 V/cm in 0.5X TBE (50 mM Tris, 45 mM boric acid, 1.25 mM EDTA) at 4°C for 2 hr 30 min. The fraction of bound RNA (Θ) was quantitated using a PhosphorImager (Molecular Dynamics). Using Imagequant, the data were fitted by nonlinear least-squares analysis as a function of protein concentration using the equation Θ =

[11] 31w [protein] [protein] + K D where Θ is the fraction of bound RNA and K D is the dissociation constant. Each K D is the average of at least three experiments.