16 Life Science : Structural Biology Crystal structure of the pre-microRNA nuclear export machinery Mature microRNAs (miRNAs), which form a class of recently discovered non-coding RNAs, are present in a wide range of eukaryotes, fungi, plants, and animals [1]. miRNAs play important roles in the regulation of biological processes, including genetic development, cell proliferation, cell differentiation, apoptosis, transposon silencing, and antiviral defense [2]. miRNA biogenesis [3] begins in the nucleus, where capped and polyadenylated pri-miRNAs, several kilobases in length, are transcribed. The pri- miRNAs are processed by the nuclear RNase III enzyme Drosha to generate ~ 65 nt pre-miRNAs that form stem-loop structures containing 2-nt 3’ overhangs. The pre-miRNAs are translocated from the nucleus to the cytoplasm through a nuclear pore complex by exportin-5 (Exp-5) in a RanGTP dependent manner. In the cytoplasm, the premiRNAs are further processed by the cytoplasmic RNase III enzyme Dicer, which excises a ~22-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. Exp-5 facilitates miRNA biogenesis by not only acting as the nuclear export factor for pre-miRNAs but also protecting pre-miRNAs from digestion by nucleases. The loss of Exp-5 results in the loss of cytoplasmic miRNA expression without the nuclear accumulation of pre-miRNAs. Pre-miRNA binding to Exp-5 requires GTP-bound forms of the small nuclear GTPase Ran cofactor (RanGTP). The Exp-5:RanGTP:pre-miRNA heteroternary complex formed in the nucleus is exported to the cytoplasm. We have determined the X-ray crystal structure of the Exp-5:RanGTP:pre-miRNA ternary complex at a 2.9 Å resolution [4]. Structural analysis was carried out using beamline BL44XU . The crystal structure of the Exp-5:RanGTP:pre- miRNA complex, which contains full-length human Exp-5, canine RanGTP, and the human pre-miRNA- 30a stem domain including the 2-nt 3’ overhang, is shown in Fig. 1. The structure of Exp-5 contains 20 HEAT repeats, each comprising two antiparallel a -helices. The pre-miRNA-30a adopts a typical A-form RNA helical structure, 60 Å in length and 20 Å in diameter. The Exp-5:RanGTP:pre-miRNA complex is ellipsoidal with dimensions of 65 × 80 × 110 Å 3 . The Exp-5:RanGTP complex forms a baseball-mitt-like structure in which the pre-miRNA is caught (Fig. 2). A tunnel-like structure at the bottom of the mitt connects the inner space of the mitt with the outer space (Fig. 2). The double-stranded stem domain of the pre-miRNA is packed into the mitt and the 2-nt 3’ overhang is located in the tunnel (Figs. 3 and 4). The 2-nt 3’ overhang structure is a characteristic feature of pre-miRNA. The protruding unpaired 2-nt 3’ end is inserted deep into the tunnel constructed from the loop and the inner helix of HEAT12, the loop and the N-terminal end of the inner helix of HEAT13, the N-terminal region of the inner helix of HEAT14, the C-terminal half loop, and the N-terminal region of the inner helix of HEAT15. The tunnel is 15 Å in length, and its shortest diameter is 10 Å. The inner surface of the tunnel is positively charged (Figs. 2 (a) (b) Fig. 1. Structure of the Exp-5:RanGTP:pre-miRNA-30a complex. (a) The structure shows human pre-miRNA-30a (red and green) bound to Exp-5 (pink) with RanGTP (purple). The long loop of HEAT15 is shown as a blue wire. In front of this view, the pre-miRNA is shielded by the long loop of Exp-5 and RanGTP. (b) The pre-miRNA molecule is viewed from the front open side. Exp-5 covers most of the stem moiety, and the 2-nt 3’ overhang structure (circled black) has many interactions with HEAT12–15, each of which is given a letter “H” with a corresponding number. 17 and 4(b)) and probably stabilizes the negatively charged 2-nt 3’ overhang structure. 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 Fig. 4. Because all interactions involve atoms of the sugar- phosphate backbone, 2-nt 3’ overhang recognition by Exp-5 is independent of the RNA sequence. Although Exp-5 is an acidic protein with pI= 5.6, it has localized positive charges from basic residues on the inner surface of the mitt that interact with a negatively charged double-stranded RNA (Fig. 2). The interacting residues of Exp-5 are distributed broadly on the inner helices of HEAT6-19 and a loop of HEAT15 to recognize the outer phosphodiester group of the pre-miRNA stem (Fig. 3). Thus, the stem of the pre-microRNA, 45 Å in length, is roughly recognized through a broad range of positively charged inner surface residues of the Exp-5:RanGTP mitt. Since both 3’ and 5’ ends of the pre-miRNA are completely shielded in the tunnel, the pre-miRNA is protected from digestion by exonucleases. Exp- 5:RanGTP surrounds the pre-miRNA on four sides, protecting it from ribonuclease digestion during export from the nucleus to the cytoplasm. Thus, Exp-5:RanGTP may act as both a nuclear export carrier and a molecular stabilizer of pre-miRNAs. (a) (b) Fig. 4. Structure of the 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 shown in pink for C a , in deep blue for N, in red for O, and in sky blue for C of the side chain. RNA is shown in deep blue for N, in red for O, in yellow for P, and in gray for C. (b) Electrostatic potential is represented as in Fig. 2. Fig. 3. Intermolecular interactions in the Exp-5: RanGTP:pre-miRNA-30a complex are shown schematically. The pre-miRNA is shown in red and light brown, where red regions interact with more basic residues of Exp-5 than light brown regions. A pink double octagon represents each HEAT repeat. 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. 2. Electrostatic surface potentials of Exp-5:RanGTP. The potentials are represented in a color gradient from red to blue for the vertex with potentials from –0.1 to 0.1 V. The black backbone represents the stem moiety of the pre-miRNA. Eiki Yamashita a , Chimari Okada-Jiko b and Tomitake Tsukihara a,c, * a Institute for Protein Research, Osaka University b Career Path Promotion Unit for Young Life Scientists, Kyoto University c Department of Life Science, University of Hyogo *E-mail: tsuki@protein.osaka-u.ac.jp References [1] R.C. Lee et al. : Cell 75 (1993) 843. [2] V. Ambros: Nature 431 (2004) 350. [3] V.N. Kim et al. : Nat. Rev. Mol. Cell Biol. 10 (2009) 126. [4] C. Okada, E. Yamashita, S.J. Lee, S. Shibata, J. Katahira, A. Nakagawa, Y Yoneda, T. Tsukihara: Science 326 (2009) 1275.
home
- JP
- EN