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A High-Resolution Structure of the Pre-microRNA Nuclear Export Machinery

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Science  27 Nov 2009:
Vol. 326, Issue 5957, pp. 1275-1279
DOI: 10.1126/science.1178705

Pre-MicroRNA Export Machinery

Micro (mi) RNAs play a role in the regulation of many biological processes. Long transcripts are initially processed in the nucleus to yield pre-miRNAs that are translocated through the nuclear pore complex and further processed to mature miRNAs in the cytoplasm. Okada et al. (p. 1275; see the Perspective by Stewart) describe the crystal structure of pre-miRNA complexed with the exportin Exp5 and the small nuclear GTPase RanGTP. The structure shows that Exp5 and RanGTP protect the miRNA from degradation by nucleases, as well as facilitate transport to the cytoplasm. RNA recognition is mainly through ionic interactions that are sequence independent, and model-building suggests that this nuclear export machinery could accommodate other small-structured RNAs.

Abstract

Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)–bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3′ overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt–like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3′ overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.

Mature microRNAs (miRNAs), short noncoding RNAs present in a wide range of eukaryotes (1, 2), play important roles in the regulation of biological processes including development, cell proliferation, cell differentiation, apoptosis, transposon silencing, and antiviral defense (36). miRNA biogenesis (7) begins in the nucleus, where capped and polyadenylated primary miRNAs, several kilobases in length, are transcribed. These are processed by the nuclear ribonuclease (RNase) III enzyme Drosha to generate ~65-nucleotide (nt) pre-miRNAs that have stem-loop structures containing 2-nt 3′ overhangs. Exp-5 translocates pre-miRNAs from the nucleus to the cytoplasm through the nuclear pore complex (812). In the cytoplasm, the pre-miRNAs are further processed by the cytoplasmic RNase III enzyme Dicer, which excises a ~22–base pair (bp) RNA duplex. One strand of the duplex binds to its target mRNA with imperfect complementarity, usually within the target’s 3′ untranslated region, assisted by the RNA-induced silencing complex (7).

Exp-5 facilitates miRNA biogenesis not only by acting as the nuclear export factor for pre-miRNAs but also by protecting pre-miRNAs from digestion by nucleases. Loss of Exp-5 results in the loss of cytoplasmic miRNA expression without nuclear accumulation of pre-miRNAs (10). Pre-miRNA binding to Exp-5 requires the guanine triphosphatase (GTPase) Ran (RanGTP). The Exp-5:RanGTP:pre-miRNA heteroternary complex formed in the nucleus is exported to the cytoplasm. Ran GTPase–activating protein, which promotes guanine triphosphate (GTP) hydrolysis in conjunction with RanBP1 and/or RanBP2, is exclusively localized in the cytoplasm and triggers the conformation change of Ran to induce release of the pre-miRNA cargo from Exp-5 (13, 14).

Here, we report the structure of the Exp-5:RanGTP:pre-miRNA complex at 2.9-Å resolution (Fig. 1A and fig. S1). This complex contains full-length human Exp-5, canine RanGTP residues 1 to 176 (removal of residues 177 to 216 stabilizes the GTP-bound conformation), and the 48-nt human pre-miRNA-30a stem domain, which includes the 2-nt 3′ overhang (nucleotide numbers 1 to 24 and 40 to 63 of human pre-miRNA-30a). Phase information used for the crystal structure analysis was derived from crystals containing Se-methionine–substituted Exp-5, and the RNA sequence was assigned from the Br anomalous signal information in crystals containing pre-miRNA 5-bromo-oxyuracil derivatives. The structure was refined to an R factor of 0.247 and free R factor of 0.312, and phasing statistics are provided in table S1. We modeled 1082 of 1204 residues of Exp-5. Several loop regions in the 20 HEAT repeats and 55 residues at the C terminus could not be modeled (details in fig. S1), and 13 residues at the C terminus were modeled as a polyalanine α helix. The residues 1 to 6 of Ran were not modeled because of their disordered structure. Electron density for the pre-miRNA was detected for nucleotides 1 to 11, 14 to 24, and 40 to 63 (fig. S2). The pre-miRNA-30a adopted a typical A-form RNA helical structure, 60 Å in length and 20 Å in diameter.

Fig. 1

The structure of the Exp-5:RanGTP:pre-miRNA-30a complex. (A) (Left) The structure shows human pre-miRNA-30a (red and green) bound to Exp-5 (pink) with RanGTP (purple). The noncrystalline portion of the pre-miRNA is represented in gray. The long loop of HEAT15 is shown as a blue wire. At the front of this view, the pre-miRNA is shielded by the long loop of Exp-5 and RanGTP. (Right) The pre-miRNA molecule is viewed from the front open side. This is the standard view presented in this paper. Exp-5 covers most of the stem moiety, and the 2-nt 3′ overhang structure (circled black) has many interactions with HEAT repeats 12 to 15, each of which is given a letter “H” with a corresponding number. (B) Electrostatic surface potentials of Exp-5:RanGTP calculated by using eF-site (26). The potentials are represented in a color gradient from red to blue for the vertex with the potential from –0.1 V to 0.1 V. The black backbone represents the stem moiety of the pre-miRNA to facilitate the display of the pre-miRNA binding site. The inner surface of the mitt is positively charged, and no intensively acidic area is found on the inner surface of Exp-5, distinguishing it from other importin-β family proteins.

The Exp-5:RanGTP:pre-miRNA complex is an ellipsoid with dimensions of 65 Å by 80 Å by 110 Å. The crystal structure contains two ternary complexes, labeled A and B, in the asymmetric unit, which are essentially similar [root mean square (RMS) of 1.84 Å, where B is slightly more open than A] and present the same recognition modes for the pre-miRNA. Detailed structural comparison of ternary complexes A and B is described in (15). The structure of Exp-5 resembles a tightly wound spring, as seen in other members of the importin-β family. Such conformations are expected to be intrinsically flexible, so small changes in the relative orientation of successive HEAT repeats could cumulatively generate substantial changes in the helicoidal pitch (16). Ternary complex A yielded more contrast in its electron density map than did complex B; thus, all structural descriptions of the ternary complex in the following discussion will be restricted to ternary complex A. The Exp-5:RanGTP complex forms a baseball mitt–like structure in which the pre-miRNA is packed (Fig. 1B). A tunnel-like structure at the bottom of the mitt connects the inner space of the mitt with the outer space (Fig. 1B).

The pre-miRNA stem is caught in the mitt formed by the Exp-5:RanGTP complex (Fig. 1), whereas the 15-Å 2-nt 3′ overhang is inserted into a tunnel formed from elements of HEAT repeats 12 to 15 (Figs. 2 and 3 and fig. S3). The inner surface of the tunnel is positively charged (Figs. 1B and 3B) and probably stabilizes the negatively charged 2-nt 3′ overhang structure. At the tunnel entrance, located at the bottom of the mitt, the guanidyl group of Arg602 (HEAT12), which is engaged in tight π-π stacking with the base pair of G1:C61 (Fig. 3 and fig. S3), sterically blocks the double-stranded stem from entering the tunnel. The 2-nt 3′ overhang structure in the tunnel is stabilized by a number of hydrogen bonds and salt bridges with amino acid residues of Exp-5, as shown in table S2, Fig. 3, and fig. S3. Because all interactions involve atoms of the sugar-phosphate backbone, 2-nt 3′ overhang recognition by Exp-5 is independent of RNA sequence.

Fig. 2

Intermolecular interactions in the Exp-5:RanGTP:pre-miRNA-30a complex are shown schematically. The pre-miRNA is colored in red and light brown, where red regions interact with more basic residues (27) of Exp-5 than do light brown regions. A pink double octagon represents each HEAT repeat. Underlined residues interact with bases of RNA with interatomic distance shorter than 3.5 Å. Exp-5 recognizes pre-miRNA through charged residues such as Arg and Lys. Exp-5 residues shown in red are involved in the recognition of the 2-nt 3′ end of the pre-miRNA, and black residues indicate interactions with the double-stranded stem of the pre-miRNA. Green residues indicate interactions between Exp-5 and RanGTP.

Fig. 3

The structure of 2-nt 3′ overhang of the pre-miRNA in the tunnel viewed from outside of the Exp-5 molecule. (A) Hydrogen bonds or salt bridges in the tunnel are represented by broken lines. Exp-5 is colored in pink for Cα, deep blue for N, red for O, and sky blue for the other side chain atoms. RNA is colored in deep blue for N, red for O, yellow for P, and green for the other atoms. Exp-5 residues [Arg602, Thr641, Gln642, Arg718, and Arg835 (27)] have hydrogen bonds or salt bridges to pre-miRNA nucleotides (G62 and C63) with interatomic distances shorter than 3.5 Å. (B) Electrostatic potential is represented as in Fig. 1B. Inside of the tunnel is electrostatically basic and shows many hydrophilic interactions with the 2-nt 3′ overhang structure.

A previous study of the 3′ overhang structure of pre-miRNA-30a showed that mutants containing 3′ overhangs with a length between 1 and 8 nt had similar binding activity to Exp-5 (17, 18). It is probably because the three additional nucleotides of the 5-nt 3′ end would pass through the aperture to maintain the interaction between Exp-5 and the 2-nt 3′ overhang of the pre-miRNA (Figs. 1A and 3).

In contrast to the above observations concerning the 3′ end of the RNA, in vitro Exp-5 binding assay showed that mutants containing a 5′ overhang of either 2 or 5 nt have reduced binding affinity to Exp-5 (17). A model of such a mutant with a 5′ overhang built with the crystal structure as a template suggests that the 3′ end of the stem would sterically clash with HEAT 11 and 12 if the 5′ overhang were to be inserted into the tunnel (Fig. 4). The modeling thus suggests how Exp-5 might discriminate between 3′ and 5′ end structures.

Fig. 4

The predicted model of a 2-nt 5′ overhang double-stranded RNA (top left) is depicted in the same orientation as Exp-5:RanGTP:pre-miRNA (top right). The model of 2-nt 5′ overhang double-stranded RNA is colored in green for its 5′ overhang region and red for the other part. Their 5′ end and 3′ end in the tunnel are depicted in surface representations viewed from outside of the mitt (bottom left and bottom right, respectively). Surface was calculated by a program MSMS (28). The 2-nt 5′ overhang is inserted into and the 3′ end of the stem sterically clashes with HEAT 11 and 12 in the predicted structure. The clashed regions are highlighted by the green dotted circle.

Although Exp-5 is an acidic protein with isoelectric point (pI) = 5.6, the protein has localized positive charges from 27 basic residues on the inner surface of the mitt that interact with the negatively charged double-stranded RNA (dsRNA) (Fig. 1B). This contrasts with the related importin-β, which has a negatively charged inner surface to accept the basic IBB domain of importin-α (19) (fig. S4). Of the 27 basic residues on the inner surface of the mitt, 11 arginine residues, 1 lysine residue, and 1 histidine residue are involved in 21 interactions with the 16-bp stem of the pre-miRNA molecule within an interatomic distance of 4 Å, as listed in table S3. The interacting residues of Exp-5 are distributed broadly on the inner helices of HEAT 6 to 19 and a loop of HEAT15 to recognize the outer phosphodiester group of the pre-miRNA 16-bp stem (Fig. 2 and fig. S5). By interacting with phosphate groups of the double-stranded pre-miRNA, Lys132 and Lys134 of RanGTP bridge phosphate groups of a major groove. The hydrogen bond between Glu445 carboxyl group and guanine base of G55 is one of two hydrogen bonds between amino acid side chains and bases in the double-stranded stem of the pre-miRNA. It does not appear to be base-specific, as judged by the hydrogen bond geometry. The dipole moment of the inner helix of HEAT19 is attracted to the negative charge of the backbone (fig. S5), as occurs in Xenopus laevis RNA-binding protein A (20). The interatomic distances between the RNA stem and the proteins are significantly longer than those within the tunnel region, and the number of contacts per nucleotide in the mitt is notably fewer than those in the tunnel (table S3 and fig. S5). Thus, the 16-bp stem of the pre-miRNA, 45 Å in length, is most likely roughly recognized through a broad range of positively charged inner surface residues of the Exp-5:RanGTP mitt.

This structural study indicates that pre-miRNA interacts with Exp-5:RanGTP mainly through ionic interactions in an RNA sequence–independent manner. This is consistent with an experiment that shows that a high ionic strength medium [containing 50 mM MgCl2 at pH = 7.4, 20 mM tris-HCl, and 1 mM dithiothreitol (DTT)] releases pre-miRNA from the Exp-5:RanGTP:Pre-miRNA ternary complex. RNA sequence–independent recognition by Exp-5:RanGTP is supported by in vitro binding assays, which demonstrated that the binding of pre-miRNA to Exp-5 occurs through the double-stranded stem and protruded 2-nt 3′ overhang but does so independently of RNA sequence (17).

Because both 3′ and 5′ ends of the pre-miRNA are completely shielded in the tunnel, the pre-miRNA is protected from digestion by exonucleases.The structure of Exp-5:RanGTP:pre-miRNA indicates that Exp-5:RanGTP prevents an endonuclease from approaching the pre-miRNA in the ternary complex (Fig. 1 and fig. S6). Exp-5:RanGTP surrounds the pre-miRNA on four sides, protecting it from ribonuclease digestion during export from the nucleus to the cytoplasm. Consistent with this, cytoplasmic miRNA expression is controlled by Exp-5 expression, and low Exp-5 expression leads to nuclear degradation of pre-miRNAs (10). Thus, Exp-5:RanGTP may act as both a nuclear export carrier and a molecular stabilizer for pre-miRNAs.

It is known that Exp-5 exports not only pre-miRNAs but also other small structured RNAs, such as tRNAs, human Y1 RNA, and adenovirus VA1 RNA, all of which have 3′ overhang structures (18, 21). Formation of the corresponding Exp-5:RanGTP:RNA complexes was examined by building structural models of the ternary complexes for these RNAs with use of the ternary complex of Exp-5:RanGTP:pre-miRNA as a template. These small RNAs could be efficiently packed into both the basic inner surface of the mitt and the tunnel. In the resulting models, the tunnel exhibiting highest positive charge of the molecule recognizes a 3′ overhang structure carrying negative charge; the inner basic surface of the mitt surrounds the sugar-phosphate backbone of double-stranded stem moieties; and lastly, the mitt accommodates small protrusions at the center of the stem in the mitt by low-energy conformational changes of Exp-5 created by a springlike movement. Detailed structure predictions are described in the SOM. A structure of Exportin (Xpot) from Schizosaccharomyces pombe in complex with RanGTP and tRNA was recently reported by Cook et al. (22). No sequence homology was detected between Exp-5 and Xpot, and Xpot does not exhibit a structure corresponding to the tunnel of Exp-5 that recognizes the 3′ overhang structure. In addition, the orientation of the RNA stem in the Xpot:RanGTP complex is inverse to that in Exp-5:RanGTP.

In radiolabel binding studies of Exp-5:RanGTP, five times more human Y1 RNA than VA1 RNA was required to compete for complex formation (18). This may be due to the fact that VA1 has only four single-nucleotide protrusions, so the larger protrusion in human Y1 may explain the inefficiency of the Exp-5:RanGTP:RNA complex formation compared with that of VA1 resulting from steric hindrance.

The truncated RanGTP superimposed well on the corresponding region of full-length RanGTP [Protein Data Bank (PDB) under accession code 1IBR] with an RMS deviation of 0.44 Å for the Cα atoms. Exp-5 interacts with RanGTP at three sites, and the detailed interaction is shown in Fig. 2. The first Ran binding site of Exp-5 is formed by HEAT repeats 1 to 5 and wraps around the Ran switch II loop similarly to other importin-β family molecules (23, 24). The second Ran-binding interface of Exp-5 is formed by HEAT repeats 7 and 8 and is similar to the binding interface of a yeast importin-β (Kap95p) and CRM1 (25). The third Ran-binding interface of Exp-5 is formed by HEAT repeats 17 and 19, which is different from those of other published importin-β family proteins (2325). The total buried surface between Exp-5 and RanGTP (1725 Å2) is the smallest of all previously reported RanGTP-binding complexes: The buried surface in the Cse1:RanGTP:Kap60p structure was 1822 Å2, that of yeast importin-β Kap95p:RanGTP was 2044 Å2, and that of CRM1:RanGTP:Snurportin1 was 2227 Å2. The smallest buried surface of Exp-5 among the importin-β family is primarily due to the difference in the third interaction site. The few contacts between RanGTP and HEAT repeats 17 and 19 might contribute to the relatively high flexibility of the mitt structure.

Supporting Online Material

www.sciencemag.org/cgi/content/full/326/5957/1275/DC1

Materials and Methods

Figs. S1 to S11

Tables S1 to S3

References

  • * These authors contributed equally to this work.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  3. The URL of the MSMS program is www.scripps.edu/~sanner/html/msms_home.html.
  4. This work was supported in part by grants-in-aid for scientific research (16087101, 16087206, and 21227003), the GCOE program (A-041) from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan (to T.T.), the Strategic Japan-UK Cooperation Program of the Japan Science and Technology Agency (to T.T.), a grant from Takeda Science Foundation (to Y.Y.), and a grant from Chungbuk BIT Research-Oriented University Consortium (to S.J.L.). Coordinates and structure factures have been deposited to the PDB under accession code 3A6P.
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