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

Supporting Online Material for

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

Chimari Okada, Eiki Yamashita, Soo Jae Lee,* Satoshi Shibata, Jun Katahira, Atsushi Nakagawa, Yoshihiro Yoneda,* Tomitake Tsukihara*

*To whom correspondence should be addressed. E-mail: sjlee@chungbuk.ac.kr (S.J.L.);

yyoneda@anat3.med.osaka-u.ac.jp (Y.Y.); tsuki@protein.osaka-u.ac.jp (T.T.)

Published 27 November 2009, Science 326, 1275 (2009) DOI: 10.1126/science.1178705

This PDF file includes:

Materials and Methods Figs. S1 to S11 Tables S1 to S3 References

2

Okada,Yamashita and Lee et al. Materials and methods Expression and purification of Exp-5 Full-length human Exp-5 (residues 1 to 1204) was cloned into the

histidine-tagged pQE60 vector in E. coli strain M15(pREP4) cells (Qiagen). The subcloned cells were grown in LB medium supplemented with 10% glycerol and were overexpressed at 20°C. The cells were suspended in buffer A (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 10 mM MgCl2, 5% glycerol, 10 mM β-mercaptoethanol, 10 mM imidazol, 1 mM Pefabloc) and disrupted by sonication on ice. Clarified lysates were loaded onto Ni-NTA agarose (Qiagen), washed with buffer A, and eluted with buffer A containing 80 mM imidazole. After incubating the Ni-NTA eluent with phenyl sepharose CL-4B (GE healthcare) for 30 min at room temperature, the beads were washed with buffer B (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 5% glycerol, 2 mM DTT) and eluted with MilliQ (Millipore). After the above preparation procedures, no significant bands other than Exp-5 were detected by SDS-PAGE analysis. Expression and purification of the Ran deletion mutant The coding sequence for the C-terminal deletion mutant of canine Ran (residues 1–176) was subcloned into a Glutathione-S-transferase (GST) gene fusion pGEX-TEV vector in E.coli strain BL21(DE3) pLysS (Stratagene) and expressed overnight at 20°C. After inducing protein expression, the cells were suspended in buffer C (50 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 5% glycerol, 2 mM DTT, 1 mM Pefabloc) and lysed by sonication on ice. Clarified lysates were loaded onto Glutathione Sepharose-4B beads (GE healthcare) for 1 h and washed with buffer C. Purification of Exp-5:RanGTP:pre-miRNA ternary complex After binding of Ran proteins to GS-4B beads, purified Exp5 and

synthesized double-stranded pre-miRNA (nucleotide numbers 1 to 24 and 40 to 63 of the human pre-miRNA30a) were incubated for 2.5 h in the presence of 0.1 mM GTP and RNase inhibitor (Promega) in buffer C. After the binding reaction was complete, beads were washed and eluted with buffer C containing 10 mM glutathione. The purified Exp5:RanGTP:pre-miRNA

3

Okada,Yamashita and Lee et al. Materials and methods complex was loaded onto Ni-NTA agarose for 1 h, washed with buffer D (50

mM Tris-HCl, pH 7.4, 2 mM MgCl2, 5% glycerol, 10 mM β-mercaptoethanol, 10 mM imidazol), and eluted with buffer C containing 80 mM imidazole. This preparation step ensured that complex formation proceeded correctly, yielding native ternary complexes. The GST-tag was cleaved overnight with 400 U of TEV protease (Invitrogen) in a total volume of 7 ml and purified by superdex200 26/60 (GE healthcare) in 20 mM Tris-HCl (pH7.4) 2 mM MgCl2, 2 mM DTT. The complex was concentrated to 7.5 mg/ml using a Millipore concentrator (Mr cut 5,000). Crystallization The Exp-5:RanGTP:pre-miRNA complex was crystallized at 293 K by the

hanging-drop vapor diffusion technique. Hanging drops (2 µl) were prepared by mixing 1 µl of the protein complex (containing 0.04 mM truncated pre-miRNA, 50 mM DTT, and 2 mM spermine tetrahydrochloride) and 1 µl of reservoir solution (containing 50 mM MES buffer, pH 6.5, 50 mM DTT, 2 mM MgCl2, 2 mM spermine tetrahydrochloride, 6.5-8% PEG3350). Plate-shaped single crystals of Exp-5:RanGTP:pre-miRNA, typically 600×200×30 µm, were obtained by streak seeding after four days. X-ray crystallography For X–ray diffraction experiments, plate-shaped crystals of the

Exp-5:RanGTP:pre-miRNA ternary complex were transiently soaked in the corresponding reservoir solutions, and PEG400 concentration was gradually increased to 25% by 5% increments accompanied by flash-cooling in a nitrogen stream at 100 K. The crystal belongs to the space group P21 with unit cell dimensions a=71.1, b=304.6, c=89.2 (Å). The Native data (λ=0.9000 Å), the Se-MAD data (remote= 0.9642 Å, edge= 0.9794 Å, peak= 0.9791 Å), and the Br peak data (peak= 0.9194 Å) sets were collected at 90 K at the SPring-8 beamline, BL44XU, Hyogo, Japan. All data were processed with the HKL2000 software package (S1). MAD phasing with 72 of the 92 possible selenium sites of Exp-5 molecules

was carried out using the SHARP software package (S2). Phases were

4

Okada,Yamashita and Lee et al. Materials and methods refined by solvent flattening and histogram matching using DM from the CCP4 program suite. An electron density map, referred to as the DM map, was calculated from the phases using the program DM. An anomalous difference Fourier map was calculated with coefficients of {F (h k l) – F (-h -k -l)} exp{i(α-π/2)} for the Se-MAD (peak) data. Of 46 Met sites in the A molecule, 39 Se sites are clearly shown in the map (fig. S9), while 33 Met sites of the B molecule were located in the map. Met residues at 1, 3, 956, 998, 999, 1008, and 1185 of A molecule are not located in the map because they reside in disordered loop regions. Model building and refinement were achieved using the programs O, CNS, and REFMAC5 (S3-S5) that was applied for the later stage of the refinement. An initial model of the ternary complex was built in the DM map. Composite omit maps and the DM map were used for further model revision to reduce the effect of a local incorrect structure. Four regions of the DM map with structural models are depicted in figs. S10A, B ,C and D. Anomalous difference Fourier maps were calculated for crystals containing pre-miRNA 5-bromo-oxyuracil derivatives to assign the RNA sequence (fig. S9). After several iterations of model revision and structural refinement, the refinement converged to Rwork of 24.7% and Rfree of 31.2%. The quality and stereochemistry of the refined structure was evaluated using the program PROCHECK of CCP4. 88.7%, 10.0%, 1.0%, and 0.3% of amino acid residues are found in the most favored region, additional allowed region, generously allowed region, and disallowed region, respectively. No inter-molecular contact shorter than 2.2 Å was detected in the crystal structure. A summary of the crystallographic data and refinement statistics is given in table S1.

5

Okada,Yamashita and Lee et al. Supporting structures Two ternary complexes in the asymmetric unit The two ternary complexes in the asymmetric unit presented the same

mode of recognition of the pre-miRNA. The average r.m.s. deviation of Cα atoms in the Exp-5 proteins in complexes A and B was 1.84 Å, including Cα atoms of the entire Exp-5 (fig. S11). Ternary complex A yielded more contrast in its electron density map than complex B, and the averaged B-factor of complex A was significantly lower than that of complex B (table S1). The baseball mitt-like structure of Exp-5 in the complex B was more open than that of the complex A by 5 Å at an edge of the mitt (fig. S11). Complex A had 121 interactions in the complex, whereas complex B interacted within the complex at 88 sites. The Exp-5 is a tightly wound spring, as seen in other members of the importin-β family, is expected to be intrinsically flexible (16), so the difference in crystal packing may affect structural difference between two complexes. Structural features of the pre-miRNA in the ternary complex Although the double-stranded 19 to 24:40 to 45 RNA could be modeled into

the electron density, this region was characterized by high atomic temperature factors and was more flexible than the other portions of the double-stranded stem. Increased flexibility arises most likely because the bottom region of the stem, composed of residues 1 to 11 and 14 to 18:46 to 61, is held in the mitt of the Exp-5:RanGTP complex, while the top region consisting of residues 19 to 24:40 to 45 is positioned outside of the mitt and has no interaction with Exp-5:RanGTP. In other words, the top region, stem 19 to 24:40 to 45, and the terminal loop region of pre-miRNA are not essential for binding to Exp-5:RanGTP. A pre-miRNA with a loop structure attached to the end of a 22 bp stem would most likely be acceptable as cargo for the Exp-5:RanGTP complex because the loop region (shown in grey in fig. 1A), positioned in the open space of the Exp-5:RanGTP complex, would not perturb formation of the Exp-5:RanGTP:pre-miRNA complex.

6

Okada,Yamashita and Lee et al. Supporting structures Structural predictions of the Exp-5:RanGTP:small RNA complexes based on the structure of Exp-5:RanGTP:pre-miRNA Exp-5 exports not only pre-miRNAs but also other small structured RNAs,

including tRNAs, human Y1 RNA, and adenovirus VA1 RNA (18, 21). Formation of the Exp-5:RanGTP:RNA complexes with these small RNAs was examined by building structural models of the ternary complexes using the ternary complex of Exp-5:RanGTP:pre-miRNA as a template. The initial structural models of Exp-5:RanGTP:RNAs were built by superposing double-stranded stems of RNAs on the stem of the pre-miRNA in the Exp-5:RanGTP:pre-miRNA complex and were refined by using a program REFMAC to reduce inter-molecular clash. The structural model of Exp-5:RanGTP:tRNA complex shows that a tRNA

with a 3’ overhang structure (PDB code 1ehz) is efficiently packed into both the basic inner surface of the Exp-5:RanGTP mitt and tunnel. Although tRNAs have a D loop protrusion and a large protrusion including an anticodon loop at the center of the 45 Å double-stranded stem, and a T loop at an end of the stem, these protrusions do not disturb tRNA binding to the complex because the D loop is accommodated in a vacant space of the mitt, the large anticodon protrusion is in the open space at the front side of the mitt and the T loop is in the open space at top of the mitt (figs. S7A, S7B and S8A). Adenovirus VA1 RNA, with a 5-nt 3’ overhang structure, contains an

A-form double-stranded stem consisting of 22 bp with two single-point nucleotide mismatches (fig. S8B). The structural model of the Exp-5:RanGTP:adenovirus VA1 RNA complex is shown in fig. S7B. Two nucleotides of the 5-nt 3’ overhang are located in the tunnel, while the remaining nucleotides of the 3’ end pass through the tunnel. Sixteen base pairs of the ~45 Å stem are accommodated in the mitt without any clash, as in the case of the pre-miRNA. The other portion of this RNA remains outside of the Exp-5:RanGTP complex. In vitro nuclear export experiments show that VA1 RNAs with more than 14 bp in the terminal stem and a 3–8 nt 3’ overhang structure (18) are bound to Exp-5:RanGTP and are exported from the nucleus to the cytoplasm (17). These results agree well with the current

7

Okada,Yamashita and Lee et al. Supporting structures structural study, since a longer stem and a longer overhang would just protrude

further from the mitt and through the tunnel. The structural model of the Exp-5:RanGTP:human Y1 RNA complex is

shown in fig. S7C. Human Y1 RNA is composed of a straight 55 Å cylindrical structure with a 3-nt 3’ overhang structure and three protruded mismatches, two 5’ single-nucleotide mismatches, and a 3’ 4-nt mismatch at the center of the cylinder. The 3-nt 3’ overhang structure would most likely have many hydrogen bonds or salt bridges with the tunnel of Exp-5 as the 2-nt 3’ overhang of the pre-miRNA. Although the cylindrical region, with the protrusion, is packed into the mitt, the 4-nt protrusion clash with HEAT17–18 with a short inter-atomic distance of ~2 Å. When a structural model of the Exp-5:RanGTP:human Y1 RNA complex is built using the Exp-5:RanGTP:pre-miRNA complex B structure, in which Exp-5 has more open conformation than that in complex A, the Y1 RNA is more appropriately accommodated into the Exp-5 mitt without steric clash. The mitt of the B complex is 4-5 Å wider than that of the complex A in the region of HEAT17–18 when the equivalent interatomic distances of the complexes A and B are compared, as shown in fig. S11. The expansion caused by a spring-like movement could allow the mitt to widen to accept the Y1 RNA with a protruded bulge.

References and Notes to Materials and Methods

S1. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307-326 (1997).

S2. E. L. Fortelle, G. Bricogne, Methods in Enzymology 276, 472-494 (1998).

S3. T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallogr A 47, 110 (1991).

S4. A. T. Brunger et al., Acta Crystallogr D Biol Crystallogr 54, 905-921 (1998). S5. M. D. Winn, G. N. Murshudov, M. Z. Papiz, Methods Enzymol 374, 300-21 (2003).

8

Okada,Yamashita and Lee et al. Supporting figures A B

fig. S1. (A) The stereo pair of the Exp-5:RanGTP:pre-miRNA-30a complex. Human pre-miRNA-30a

(black, wired) bound to the 20 HEAT repeats of Exp-5 (deep pink for outer helix and sky blue for inner

helix as in fig. S1B) in complex with RanGTP (purple). HEAT20 has no inner helix. (B) The sequence of

Exp-5 and the corresponding secondary structure are represented. Residues in helical conformations

are shown in light blue (inner helix) and red (outer helix), and the long loop in HEAT15 is shown in

deep pink. Residues whose atomic coordinates are left unkown are in grey. The N-terminal residue 1,

residues 474-490 in the loop of HEAT12, residues 705-706 in the loop of HEAT14, residues 938-951 in

the loop of HEAT17 and residues 980-1069 in the loop between HEAT17 and HEAT18 are disordered

in the crystal. Out of 68 C-terminal residues, 55 residues were not modeled because of disordered

structure, and 13 residues were built as a poly-alanine α-helix because of high temperature factor.

9

Okada,Yamashita and Lee et al. Supporting figures

fig. S2 Schematic drawings of the binding modes of the Exp-5:RanGTP:pre-miRNA-30a complex. Exp-5

residues are shown in black and RanGTP residues are shown in green. The amino acids interacting

with the RNA are shown by single letter notation with sequence number. The underlined amino acid

name shows a residue having a hydrogen bond with a base. Bold nucleotides indicate double-stranded

stems and the 2-nt 3’ overhang that were resolvable in the crystal structure, whereas grey nucleotides

indicate loop or mismatched regions of the RNA that were not included in the model. Many

interactions occur only at the 2-nt 3’ end. Other binding interactions are individually weak but

collectively strong because they are distributed broadly across the RNA surface.

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Okada,Yamashita and Lee et al. Supporting figures

fig. S3. The 2-nt 3’ overhang of the pre-miRNA and the tunnel of Exp-5 are depicted in a stereo pair

viewed from outside of the Exp-5 molecule. Shown are a Cα trace of Exp-5 (pink) and residues (Arg602,

Thr641, Gln642, Arg718, and Arg835) that have hydrogen bonds or salt bridges to pre-miRNA

nucleotides G62 and C63 with inter-atomic distances shorter than 3.5 Å. Black lines show the

backbones of the pre-miRNA. Ball-and-stick models represent the amino acid residues and nucleotides.

The oxygen and nitrogen atoms are red and deep blue spheres, respectively. Bonds of amino acids and

nucleotides are represented by light blue and green sticks, respectively. Nucleotides 62 and 63 have

eight hydrogen or ionic bonds (black broken line) with the Exp-5 in the tunnel. A guanydyl group of

Arg602 is stacked on the base pair of G1:C61.

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Okada,Yamashita and Lee et al. Supporting figures fig. S4. Electrostatic surface model of importin-β. The electrostatic potentials were 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 -0.1V to 0.1V as in fig. 1B. 77.3% of molecular surface exhibit negative potential and

25.9 % of the surface were lower than -0.1 V. 22.7 % and 4.8 % of the surface have positive potential

higher than 0.0 V and 0.1 V, respectively. Highest and lowest potentials of the vertexes are 1.06 V and

-1.14 V, respectively. Acidic charges are widely distributed on the inner surface of importin-β.

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Okada,Yamashita and Lee et al. Supporting figures

fig. S5. Interaction sites between the pre-miRNA and the Exp-5:RanGTP complex in the tunnel or mitt.

For simplicity, the Exp-5 and RanGTP residues that have hydrogen bonds or ionic interactions to the

pre-miRNA with interatomic distances shorter than 4.0 Å are shown. Nitrogen and oxygen atoms of

amino acid side chains and RNA are colored in deep blue and red, respectively. The other atoms of

Exp-5, RanGTP and RNA are colored in pink, purple and green, respectively.

The inner helix of HEAT9 is kinked at the helix center to allow the strand to approach a minor groove

of the double-stranded pre-miRNA in a parallel orientation . A similar parallel orientation has been

detected in the Xenopus laevis RNA-binding protein A (20). No other helix of Exp-5 provides such close

contacts with minor or major grooves of the double-stranded stem of the pre-miRNA. The kink orients

Glu445 in HEAT9 to the G55 guanine base. The amino terminal end of the inner helix of HEAT19 is

directed toward the sugar-phosphate backbone of the double-stranded stem of the pre-miRNA. The

acidic Asp1087 residue of HEAT19 is hydrogen bonded to an oxygen atom of a sugar group.

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Okada,Yamashita and Lee et al. Supporting figures

fig. S6. A predicted model of RNase III binding to the pre-miRNA. This complex structure was

produced by superposing a double stranded RNA of RNase III:RNA complex on the pre-miRNA of

Exp-5:RanGTP:pre-miRNA.

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Okada,Yamashita and Lee et al. Supporting figures A B C D fig. S7. Predicted models of other small RNAs bound to Exp-5 (pink):RanGTP (purple). Stereoscopic

images of predicted models of Exp-5(pink):RanGTP(purple):small RNA(red) complexes. (A) A front

view of Exp-5:RanGTP:tRNA. (B) A side view of Exp-5:RanGTP:tRNA. (C) Front view of

Exp-5:RanGTP:adenovirus VA1 RNA. (D) Front view of Exp-5:RanGTP:Human Y1 RNA.

15

Okada,Yamashita and Lee et al. Supporting figures A B C fig. S8 Secondary structures of small RNAs bound to Exp-5. (A) tRNA, (B) Human Y1 RNA, and (C)

adenovirus VA1 RNA.

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Okada,Yamashita and Lee et al. Supporting figures fig. S9. Anomalous difference Fourier maps for Se-Met and Br derivatives. Contours colored in blue are

the anomalous difference peaks for Se-Met Exp-5 data (peak) at 3.8 Å resolution. Contours are drawn

at 4.5 σ. Se positions were successfully applied for the initial phase determination. Contours in pink

and yellow are for Br-derivatives of the 5’ and 3’ end strands of the pre-miRNA, respectively. They

were calculated at 4 Å resolution and drawn at 4.5 σ. The 5’ end Br-derivative was replaced by

brominated uracil at nucleotides 3, 9, 12, 17 and 24. Three anomalous difference peaks at 3, 9 and 17

were clearly detected in the map. The bromine peaks for 12 and 24 did not appear in the map because

they are in the disordered region or have high temperature factors. The 3’ end Br-derivatives are

brominated at eight sites of 43, 44, 45, 49, 54, 56, 57 and 58. The anomalous peaks for bromine atoms

of 54, 56, 57, 1nd 58 are uniquely located in the map. Significant peaks for 43, 44, 45 and 49 are not

detected in the map because of their high temperature factors. Consequently Br sites uniquely

restricted the RNA sequence assignment.

17

Okada,Yamashita and Lee et al. Supporting figures A B C D

18

Okada,Yamashita and Lee et al. Supporting figures fig. S10. Four regions of the DM map calculated with phases refined at 2.9 Å resolution. Contours are

drawn at 1.0 σ. Wire models of Exp-5 (pink) and RanGTP (purple), and a ball-and-stick model of the

pre-miRNA are superimposed on the map. (A) A region including nt 1-8 and 54-63 of the pre-miRNA.

(B) The tunnel (HEAT12-15) and nucleotides 62 and 63. (C) HEAT15 -19. A loop between the inner and

outer helices of HEAT17 was not modeled because it has no electron density. (D) HEAT18-20 and part

of the C-terminus. Although 13 of 68 residues of the C-terminus are in the helical structure, their

amino acid identities remain unknown. The other C-terminal residues were not modeled.

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Okada,Yamashita and Lee et al. Supporting figures

fig. S11. Conformational differences between the Exp-5 molecules in the asymmetric ternary

complexes A and B. By superposing the N-terminal structure, the A chain of Exp-5 (red) is more

compact than the B chain (green). The closed conformation in the A chain results in tighter complex

formation. Cα distances between Q332 (H7) and G1088 (H19) are 29.96 Å in the A chain and 35.12 Å in

the B chain. Cα distances between M373 (H8) and L1042 (H18) are 28.72 Å in the A chain and 33.6 Å

in the B chain. Cα distances between A434 (H9) and Q960 (H17) are 23.62 Å in the A chain and 27.69 Å

in the B chain. Cα distances between L508 (H10) and V894 (H16) are 31.6 Å in the A chain and 34.14 Å

in the B chain.

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Okada,Yamashita and Lee et al. Supporting tables table S1. Intensity data and phasing statistics

Data sets Native Exp-5-Se 5’endBr*1 3’endBr*2

Remote Edge Peak Peak Peak

Cell dimensions

a, b, c (Å), β (º) 71.1, 304.6 71.5, 303.9 71.5, 303.9 71.7, 304.4 71.0, 304.7 69.6, 303.3

89.2, 110.8 89.4, 111.1 89.4, 111,1 89.5, 111.2 89.4, 111.1 88.2, 110.1

Wavelength (Å) 0.9000 0.9642 0.9794 0.9791 0.9194 0.9194

Resolution (Å) 2.9 4.0 4.0 3.5 3.4 3.2

(3.0 - 2.9) (4.14 - 4.00) (4.14 - 4.00) (3.63 - 3.50) (3.52 - 3.40) (3.31 - 3.20)

No. reflections

Observed 270,865 106,427 106,445 284,614 616,577 679,792

(40,007) (9,926) (9,970) (27,189) (57,981) (67,283)

Unique 74,959 29,016 29,043 43,852 46,746 54,346

(7441) (2,861) (2,857) (4,351) (4,559) (5,420)

Redundancy* 3.6(3.5) 3.7 (3.5) 3.7 (3.5) 6.5 (6.2) 6.9(6.7) 6.7(6.7)

Completenes(%)# 99.6 (99.3) 99.2 (97.6) 99.3 (97.5) 99.2 (98.6) 98.3(95.2) 100.0(100.0)

I/σ(I) 14.6(1.9) 11.7 (3.6) 11.0 (3.1) 16.8 (3.8) 22.0(5.0) 23.7(5.9)

Rmerge$ 0.094 0.127 0.127 0.124 0.094 0.104

(0.380) (0.339) (0.379) (0.382) (0.357) (0.337)

Phasing

Resolution 4.5 4.5 3.8

Phasing Power (isomorphous)& 1.04 0.28

Phasing Power (anomalous) & 0.78 0.48 1.05

Number of sites 72

FOM (acentric / centric)% 0.34 / 0.22

Refinement

Resolution (Å) 2.9 Ramachandran plot(%)

R / Rfree** 0.247/0.312 Favored 88.8

rms deviation from ideal Allowed 10.0

21

Bond lengths(Å) 0.02 Generously allowed 1.0

Bond angles(˚) 1.83 Disallowed 0.3

close contacts based on crystal symmetries Average B factors (Å2) 96.7

< 2.5 Å 10 Complex A (Å2) 82.3

< 2.2 Å 0 Complex B (Å2) 97.8 Number in parentheses are given for the highest resolution shell. *Redundancy is the number of

observed reflections for each independent reflection. #Completenes in a percentage of independent

reflections observed. $Rmerge : ΣhiΣi|I(h,i) -< I(h)>|/ΣhiΣiI(h,i), where I(h,i) is the intensity value of the

ith measurement of h and is the corresponding mean value of I(h) for all I measurements; the

summation is over the reflections with I/σI larger than -3.0. &Isomorphous phasing power is rms

isomorphous difference divided by rms isomorphous residual lack of closure, and anomalous phasing

power is rms anomalous difference divided by rms anomalous residual lack of closure. %FOM is the

mean figure of merit. **R is a conventional crystallographic R factor, Σ|Fo - Fc|/Σ|Fo|, where Fo and

Fc are the observed and a calculated structure factors, respectively. Rfree is a free R factor of the

refinement evaluated for the 5 % of reflections that are excluded from the refinement. *15’endBr is

(base number 1-24)5’-GGXAAACAXCCXCGACXGGAAGCX-3’, and *23’endBr is (base number

40-63)5’-GGCXXXCAGXCGGAXGXXXGCCGC-3’, where X represents Br-uracil.

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Okada,Yamashita and Lee et al. Supporting tables table S2. Interaction between the 2-nt 3’ overhang and the Exp-5 tunnel.

residue of

Exp-5 Exp-5 HEAT

Contacted residue of

pre-miRNA Distance(Å）

R602 NH2 H12 G62 O1P 3.2

M643 SD H13 G62 O2P 3.7

R835 NH2 H15 G62 O5' 3.4

R835 NH1 H15 G62 O4' 3.5

R598 NH1 H12 G62 O4' 3.7

R835 NH1 H15 G62 N9 3.9

R598 NH1 H15 G62 N9 4.0

R835 NH1 H15 G62 N7 3.9

R593 NH1 H12 G62 N2 3.8

R593 NE H12 G62 N2 4.0

R593 NH2 H12 G62 N2 3.8

T641 OG1 H13 G62 O2' 2.8

T641 OG1 H13 G62 O3' 3.7

Q642 N H13 C63 O2P 2.7

Q642 OE1 H13 C63 O2P 3.9

M643 N H13 C63 O2P 3.6

T641 OG1 H13 C63 O2P 3.9

R718 NE H14 C63 O2 4.0

R718 NH1 H14 C63 O2 2.9

R835 O H15 C63 O2 3.3

G715 O H15 C63 N3 4.0

E711 OE2 H14 C63 N3 3.8

R718 NH1 H14 C63 N3 3.2

E711 OE2 H14 C63 N4 3.4

R835 O H15 C63 O2' 3.0

F839 N H15 C63 O2' 3.7

R835 NE H15 C63 O2' 3.7

R835 NH1 H15 C63 O2' 3.9

R835 NH2 H15 C63 O2' 3.7

F839 N H15 C63 O3' 3.8

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Okada,Yamashita and Lee et al. Supporting tables table S3. Interactions between the ds stem of the pre-miRNA and the Exp-5:RanGTP:complex

residue of

Exp-5 Exp-5 HEAT

Contacted residue of

pre-miRNA Distance(Å）

S606 OG H12 G1 O4' 3.8

S606 O H12 G1 O4' 3.5

S606 OG H12 G1 N9 3.0

S606 OG H12 G1 N7 3.4

S606 OG H12 G1 N3 4.0

R734 NH2 H14 G1 O2' 3.1

R734 NH2 H14 G1 O3' 3.2

R734 NH1 H14 G2 O1P 3.9

R734 NH2 H14 G2 O1P 3.6

R613 NH1 H12 G2 O1P 3.7

R893 NE H16 A4 O1P 3.5

R893 NH1 H16 A4 O1P 4.0

E445 OE1 H9 A8 O4' 4.0

E445 OE2 H9 A8 N3 3.6

E445 OE1 H9 A8 O2' 3.7

R380 NE H8 A9 O2' 3.9

D1087 OD2 H19 U17 O2' 3.5

D1087 OD1 H19 U17 O2' 3.4

G279 O H6 U45 O2' 3.8

K278 NZ H6 A47 O2P 4.0

Q1045 OE1 H18 U49 O2' 2.4

Q1045 NE2 H18 U49 O2' 4.0

H1086 NE2 H19 U49 O2' 3.9

H1086 NE2 H19 U49 O3' 3.9

R1046 NE H18 G51 O2P 3.9

R1046 NH1 H18 G51 O2P 3.9

Q1050 NE2 H18 G51 O4' 4.0

Q1050 OE1 H18 G51 O2' 4.0

24

E445 OE2 H9 G55 N3 3.0

E445 OE1 H9 G55 N2 3.5

E445 OE2 H9 G55 N2 3.6

E445 OE2 H9 G55 O2' 3.4

E445 OE2 H9 U56 O4' 4.0

A441 O H9 U56 O2' 2.6

E445 N H9 U56 O2' 4.0

R448 NH2 H9 U56 O3' 2.7

R448 NH2 H9 U57 O1P 2.8

N437 O H9 U57 O2' 3.6

A441 N H9 U57 O2' 4.0

R440 NH1 H9 U57 O3' 3.9

R440 NH1 H9 U58 O1P 3.5

R595 NH1 H12 C60 O1P 3.7

R595 NH2 H12 C60 O1P 3.9

R838 NH1 H15 C60 O2' 3.9

R598 NH2 H12 C61 O1P 3.8

R595 NE H12 C61 O2P 3.9

R602 NH2 H12 C61 N1 3.9

R602 NE H12 C61 N3 3.7

M643 SD H13 C61 O2' 3.7