0.75 1.000.25

n = 2.1 ± 0.2Kd = 2.1 ± 1.1 μM

0.50

log10[P](log10μM)-1

0

1

2

log 10

θ/(1

-θ)

0.0 1.0 2.0 2.5 3.0 3.5 4.0 5.0 6.0 9.0

pri-miR-16-1

DGCR8M- pri-miR-16-1

a bDGCR8M (µM)

Supplementary Figure 1. The binding analysis between DGCR8M and pri-miR-16-1

(a) The binding between wild type DGCR8M (0 to 9 uM) and pri-miR-16-1 (0.25nM) was analyzed by EMSA.

(b) The Hill plot of integration data from (a) is shown. The Hill constant (n) and Kd are mean values of three independent experiments.

Crystal structure of human DGCR8 core

Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho

y = -1.6294x + 7.7709R2 = 0.995

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Ve/Vo

log(

Mw

)

a b

pri-miR-16-1& DGCR8M

100 150 200 250 3000

35

70

280 nm260 nm

DGCR8M

100 150 200 250 3000

5

10

Abs

orba

nce

(mA

U)

pri-miR-16-1

100 150 200 250 3000

50

100

280 nm260 nm

280 nm260 nm

669 440 150 66 29 12.4 kDa

Elution volume (ml)

200 ml (101.8 kDa)

230ml (38.4 kDa)

204 ml (87.1 kDa)

Supplementary Figure 2. SEC analysis of the DGCR8M-pri-miR-16-1 complex (a) The standard curves for SEC experiments. The formula (indicated in figure) was derived from the linear regression curve and used to estimate the molecular weight of the DGCR8M-pri-miR-16-1 complex. The standard curves for SEC experiments were obtained from the following proteins: cytochrome C, 12.4 kDa; carbinic anhydrorase, 29 kDa; Bovin serum albumin, 66 kDa; Alcohol dehydroganase, 150 kDa; Ferritin, 440 kDa; thyroglobulin, 480 kDa. The formula was derived from the linear regression curve and used to estimate the molecular weight of the hDGCR8M-pri-miR-16-1 complex.

(b) SEC results of the DGCR8M-pri-miR-16-1 complex (top), DGCR8M (middle), and pri-miR-16-1 (bottom).

Crystal structure of human DGCR8 core

Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho

Molecular weight of DGCR8M−pri-miR-16-1 complex from equilibrium sedimentation

Concentration of

DGCR8M (mM)

Concentration of

pri-miR-16-1 (mM)

Estimated MW

(kDa)

Monomer MW of

DGCR8M (kDa)

Monomer MW of

pri-miR-16-1(kDa)

MW estimated /

MW monomer of

DGCR8M and

pri-miR-16-1

1 0.5 62.4 27 35 1.01

Model: Single ideal speciesVariance: 5.94153E-5Speed:12000 rpmTemp: 15 ℃ V-bar: 0Rho : 1MW: 62417 Da

Radius (cm)

Abo

sorb

ance

Res

idua

lsCrystal structure of human DGCR8 core

Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho

Supplementary Figure 3. Analytical ultracentrifuge analysis ofthe DGCR8M-pri-miR-16-1 complex

The equilibrium fit results of analytical ultracentrifuge for the DGCR8M-pri-miR-16-1 complex. Fitted overlay (red line) to experimental data (blue circles) is shown in the bottom panel; residuals are shown in upper panel. The fitted parameter for the weight-average molecular weight (Mwapp) was estimated at 62,417 gm-1.

5′ ag c - A C U GAUUC 3′ gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA caguug aug AGUCGUCGUG CA UUAUGACC C AUU 3′ cauac ga A U A U U AAAAT-TMR 5′

b

RNA−TMR

5′ FAM ag c - A C U GAUUC 3′ gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA caguug aug AGUCGUCGUG CA UUAUGACC C AUU 3′ cauac ga A U A U U AAAAT 5′

RNA−FAM a

5′ FAM ag c - A C U GAUUC 3′ gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA caguug aug AGUCGUCGUG CA UUAUGACC C AUU 3′ cauac ga A U A U U AAAAT-TMR 5′

RNA−FAM_TMR

Wavelength (nm)

Nor

mal

ized

fluo

resc

ence

inte

nsity

0

0.20

0.40

0.60

0.80

1.00

1.20

500 550 600 650

0.5 00.5 0.5 0.5 1.0 0.5 2.0 0.5 3.0 0.5 5.0 0.5 10.0

0.5∗ 00.5∗ 5.0

RNA−FAM_TMR (µM) DGCR8M (µM)

Wavelength (nm)

Nor

mal

ized

fluo

resc

ence

inte

nsity

0

0.20

0.40

0.60

0.80

1.00

1.20

500 550 600 650

0.5 00.5 1.0 0.5 3.0 0.5 5.0 0.5 10.0

RNA−FAM +RNA−TMR (µM) DGCR8M (µM)

Supplementary Figure 4. FRET analysis of modified pri-miRNAs forinter- or intra-molecular energy transfer

(a) A fixed amount of RNA-FAM_TMR was titrated with various concentrations of DGCR8M. RNA-FAM (donor-only labeled RNA) is marked with an asterisk.

(b) A 1:1 mixture of RNA-FAM and RNA-TMR (acceptor-only labeled RNA) was titrated with various concentrations of DGCR8M.

Crystal structure of human DGCR8 core

Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho

Protein Kd (μM) Kd (μM)

DGCR8∆N483 2.0 ± 1.4 × 10 −6 3.0 ± 1.4 × 10 −6

DGCR8L 3.1 ± 1.1 × 10 −6 4.2 ± 1.2 × 10 −6

DGCR8M 2.1 ± 1.1 × 10 −6 4.1 ± 1.3 × 10 −6

DGCR8S 2.2 ± 1.5 × 10 −6 2.9 ± 1.6 × 10 −6

5′ g ag c - A C U GAUU gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA C caguug aug AGUCGUCGUG CA UUAUGACC C AUU U 3′ ga A U A U U AAAA

pri-miR-16 ∆BS

5′ ggugauagcaau ag c - A C U GAUU gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA C caguug aug AGUCGUCGUG CA UUAUGACC C AUU U 3′ caucucauac ga A U A U U AAAA

pri-miR-16-1 WT

Supplementary Table 1. Dissociation constants for DGCR8∆N483, DGCR8L, DGCR8M and DGCR8S to the pri-miR-16-1 wild type RNA and pri-miR-16 ∆BS.

Crystal structure of human DGCR8 core

Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho

Crystal structure of human DGCR8 core

Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom,

V. Narry Kim and Yunje Cho

SUPPLEMENTARY METHODS

Protein Expression and Purification. All constructs were generated using a

standard PCR-based cloning strategy, and entire coding sequences were

verified by sequencing. The whole gene of human DCGR8 (residues 1–773)

was cloned from HEK293T cDNA, inserted into pGEX-2T vector (Amersham),

and expressed in E. coli BL21 as GST fusion protein and purified using a

glutathion Sepharose column. They were then cleaved with thrombin. Full

length DGCR8 was further purified by anion exchange and gel-filtration

chromatography. To aid crystallization, we used combination information of

limited proteolysis and sequence alignment analysis. This characterization

identified the conserved core DGCR8S (residues 493-720), which was used for

structural analysis.

His-tagged DGCR8S (residues 493-720) was also synthesized by PCR,

digested, inserted into pET28a (Novagen) vector, and transformed into E. coli

BL21 (DE3). DGCR8S was purified using a Ni-column and then subjected to

cation exchange (Mono-S) and gel-filtration chromatography (Superdex 75

column). DGCR8S used for the crystallization was first concentrated to 8 mg/ml.

DGCR8M (residues 493 to 738), DGCR8L (residues 484 to 750), and

DGCR8ΔN483 (residues 484 to 773) were cloned into pET28a and expressed

in E. coli strain BL21 (DE3). The overall purification procedure used for these

1

proteins was the same at that used for DGCR8S.

Preparation of pri-miRNA for EMSA. pri-miRNAs were prepared by in vitro

transcription. Template DNAs for pri-miR-16-1 or the pri-miR-16-ΔBS transcript

were amplified by PCR using pGEM vector containing the pri-miR-16-1 gene.

The forward primers used for PCR contained the T7 promoter sequence at their

5’ ends. The sequences of the primers used were; 5’-

TAATACGACTCACTATAGGTGATAGCAATGTCAGCAGTGCCTTAGCAG-3’

(forward primer for pri-miR-16-1), 5’-GTAGAGTATGGTCAACCTTACTTCAGCA

G-3’ (reverse primer for pri-miR-16-1), 5’-TAATACGACTCACTATAG

GTCAGCAGTG –CCTTAGCAG - 3’ (forward primer for pri-miR 16-ΔBS) and 5’-

GTCAACCTTACTTCAGCAG-3’ (reverse primer for pri-miR-16-ΔBS). The PCR

products so obtained were then used as templates for in vitro transcription to

prepare pri-miR-16-1 transcripts. Pri-miR-16-1 and pri-miR-16-ΔBS transcripts

were then purified by 8(w/v)% polyacrylamide/ 7(w/v)% urea gel electrophoresis.

Mutation, purification, and protein modification for FRET. DGCR8 mutants

were constructed using a PCR based protocol and purified using the procedure

described above for wild-type DGCR8. All five native cysteines (Cys514,

Cys535, Cys627, Cys666 and Cys675) were replaced by serine by site-directed

mutagenesis to eliminate ambiguous fluorophore labeling. Additional single site

mutations (S493C, V581C or S638C) were introduced into this mutant to create

unique cysteine residues. The Trp699 residue of mutant

(C514S/C535S/C627S/C666S/C675S/V581C) was also mutated to phenyl

2

alanine making it the only Trp donor (Trp665). Incorporations of these mutations

were confirmed by DNA sequencing.

N-(iodoacetyl)-N’-(5-sulfo-1-naphthyl) ethylenediamine (1,5-IAEDANS)

was obtained from Molecular Probes. A 10-fold molar excess of 1,5-IAEDANS

was added to DGCR8M-WC protein solution in labeling buffer containing 25mM

Tris-HCl and 200mM NaCl at pH 7.3. The labeling reaction was allowed to

proceed for 18hr at 18℃. Cysteine-labeled proteins were extensively dialyzed in

storage buffer to remove excess label.

Two DGCR8 mutants, DGCR8L-S493C and DGCR8L-S638C, were

constructed to investigate DGCR8-pri-miRNA binding modes by FRET after

labeling with Cy5 fluorophore. The modification of DGCR8M using Cy5

maleimides (Amersham) was carried out using the protocols described above.

Extents of cysteine labeling were determined by measuring AEDANS

dye concentrations at 336nm (ε=5,700 M-1·cm-1) and Cy5 dye concentrations at

650nm (ε=250,000 M-1·cm-1). Labeling efficiencies were typically >0.95 labels

per protein.

Preparation of pri-miRNA for FRET. pri-miRNAs were prepared using the

protocol described for EMSA using the following primers; 5’-

TAATACGACTCACTATAGGTGATAGCAATGTCAGCAGTGCCTTAGCAG-3’

(forward primer for the miR-16-1 Δ3’ construct) and 5’-

CACAGTTAATACTGGAGAT-3’ (reverse primer for the miR-16-1 Δ3’ construct).

Partial pri-miRNAs either labeled or unlabeled on their 5’ or 3’ ends with a

fluorophore Cy3, FAM(fluorescein) and TMR(rhodamine) purchased from

3

Samchully Pharmaceuticals. The following RNAs are synthesized; 5’-

gcugcugaaguaagguugaccauacucuac-Cy3-3’, 5’-

gcaaugucagcagugccuuagcagcacguaaauauuggcguuaagauuc-3’, 5-

aaaauuaucuccaguauuaacugugcugcugaaguaagguugaccauac-3’, 5’-FAM-

gucagcagugccuuagcagcacguaaauauuggcguuaagauuc-3’, 5-TMR-T-

aaaauuaucuccaguauuaacugugcugcugaaguaagguugaccauac-3’.

The sense-antisense pairs (RNA-FAM and RNA-FAM_TMR) were

annealed into duplexes in 1X universal buffer (6 mM HEPES-KOH, pH 7.5, 20

mM KCl, and 0.2 mM MgCl2). RNAs were denatured at 94 ºC for 1 min and

annealed by slowly (1ºC/min) decreasing temperature to 4ºC. Annealed pri-

miRNA derivatives were purified by native polyacrylamide gel electrophoresis,

recovered by soaking in elution buffer (0.5M CH3COONH4, 0.1mM EDTA, 1mM

MgCl2, 0.1 % SDS), and dialyzed against 100mM NaCl and 25mM Tris-HCl at

pH 7.3. Pri-miR-16-1 derivatives labeled with Cy3 or dual-labeled with FAM

and TMR were then used to analyze the characteristics of binding between

DGCR8 and pri-miRNA.

Fluorescence measurements and FRET calculations. Fluorescence was

measured using a Cary Eclipse Fluorescence spectrophotometer (VARIAN

Australia, Inc) equipped with a Peltier-temperature controlled sample chamber,

which was maintained at 20ºC. Steady-state fluorescence experiments were

performed with the spectrofluorimeter thermostat set at 20ºC. Fluorescence

spectrophotometer’s slits were adjusted to 5 nm. All FRET experiments were

performed over three times independently and each sample was monitored over

4

four scans.

Measurement of RNA-FAM_TMR FRET: To examine the possible RNA

conformational change, a fixed amount (0.5 μM) of FAM and TMR dual-labeled

RNA (RNA-FAM_TMR) or FAM single-labeled RNA (RNA-FAM) was titrated

with various concentrations of DGCR8M (0.5 - 10 μM). DGCR8M was added to

pri-miRNA and incubated for 5 min before measuring the fluorescence spectrum

of RNA. The samples were directly excited at 490 nm and emission signal was

collected at 500 - 650 nm during an average of four scans. The background

fluorescence caused by direct excitation of TAMRA (acceptor) at 490 nm was

measured in separate titration and subtracted. Fluorescence spectra of excess

concentration of RNA (0.5 – 2.5 μM) in the presence of a fixed concentration of

DGCR8 (0.25 μM) were also measured.

Measurement of DGCR8M-WC and DGCR8M-W_Ad: 10 μM of pri-miR-16-1

were added to same concentration of DGCR8M-WC or DGCR8M-W-Ad and

incubated for 5 min before measuring the DGCR8M derivative’s spectrum.

Fluorescence spectra were collected over a range of emission wavelengths

(λex=290 nm and λem=300–560 nm) over four scans. The background

fluorescence caused by direct excitation of AEDANS acceptor at 290 nm was

measured separately and subtracted.

Measurement the orientation of pri-miR-16-1: 0.5 μM of DGCR8L-

S493C_Cy5 or DGCR8M-S638C_Cy5 was added to a fixed concentration of

pri-miR-16-1 labeled Cy3 on 3’ end (pri-miR-16-1-Cy3, 0.2 μM) and incubated

for 5 min before measuring the spectrum. Fluorescence spectra were collected

5

over a range of emission wavelengths (λex = 550 nm, λem = 560-700 nm).

FRET calculation: The efficiency of fluorescence resonance energy transfer

(ET) was determined from donor fluorescence intensity reductions using the

following equation: ET = (1- FDA/ FD) Eq.1

Where FD and FDA are donor fluorescence intensities for donor-only and for

donor and acceptor, respectively. Measurements were performed using the

same buffer under identical concentrations, and FDA/FD ratios were averaged

over a range of 60 nm about the fluorescence maximum.

Distances between donor and acceptor were calculated from FRET

efficiencies using:

R = R0 (1/ ET-1) 1/6 Eq.2

Where R is a distance between fluorophores and R0 is the Förster distance

(defined as the distance at which energy transfer is 50 % of the maximum

value). The Foster distances (R0) between FAM and TMR, and between Cy3

and Cy5 were calculated to be 55 and 60Å, respectively 1,2.

Mutant Protein Structural Changes and Stabilities. The structural changes of

mutant proteins (10 µM) versus the wild type were monitored by circular

dichroism (CD) spectrophotometry (Jasco J-715) at wavelengths between 200-

250 nm. All samples were prepared in the buffer used for size exclusion analysis.

Size exclusion chromatography. All SEC experiments were performed using

a Hi-Load Superdex 200 26/60 column (Amersham) at 4ºC and running buffer

contained 25 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 mM DTT. To

6

characterize the stoichiometry of the DGCR8-pri-miRNA complex, pri-miR-16-1

was added to 10 molar excess concentration of DGCR8M (final concentration of

RNA: protein = 0.2: 2.0 μM) in running buffer and incubated at 4ºC for 4 hrs.

Analytical ultracentrifugation. The molecular mass of DGCR8M, pri-miR-16-1

and the DGCR8M-pri-miR-16-1 complex were analyzed by means of an

analytical ultracentrifuge optima XL-A (Beckman, PaloAlto, CA) using the

sedimentation equilibrium technique. Sedimentation equilibrium data were

evaluated using a nonlinear least-squares curvefitting algorithm (XL-A Data

Analysis Software). The value of 0.736 mlg-1 was used as the partial specific

volume. All samples were analyzed in binding buffer containing 25 mM Tris-HCl,

pH 7.5, 150 mM NaCl. For equilibrium analysis, scans at equilibrium from

multiple speeds (9,000; 12,000 and 15,000 rpm) were collected at 15ºC,

measuring absorbance at 280 nm.

7

SUPPLEMENTARY REFERENCES

1. Chapados, B.R., Hosfield, D.J., Han, S., Qiu, J., Yelent, B., Shen, B. & Tainer,

J.A. Structural Basis for FEN-1 Substrate Specificity and PCNA-Mediated

Activation in DNA Replication and Repair. Cell 116, 39-50 (2004).

2. Bowen, M.E., Weninger, K., Ernst, J., Chu, S. & Brunger, A.T. Single-

Molecule Studies of Synaptotagmin and Complexin Binding to the SNARE

Complex. Biophys. J. 89, 690–702 (2005).

8