Kawamata et al., Figure S1

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1 Kawamata et al., Figure S1 +Lysate Lysate Time (min) ss let Duplex A Duplex B ss let-7 Duplex A Duplex B Complex I Complex II Complex III Complex IV Complex V Free ighter exposure Complex V Free Supplementary Figure 1 small RNA-protein complexes formed in dcr-2 embyo lysate. Native gel analysis was performed as in Figure 1a at 25 ºC with shorter electrophoresis to trap free duplexes. Single-stranded (ss) let-7 formed complex V at the earliest time point, and then immediately degraded without forming any other complexes. Nature Structural & Molecular Biology: doi:1.138/nsmb.163

2 Kawamata et al., Figure S2 a b Passenger strand labeled Guide strand labeled Duplex A Duplex B Duplex A Duplex B Time (min) Target RNA length (nt).1k.2k 1.5k.1k.2k 1.5k Complex I Complex I Complex II Complex IV Complex IV Complex V Supplementary Figure 2 Complexes I and II represent pre-ago1-risc and mature Ago1-RISC respectively. (a) Native gel analysis was performed as in Figure 1a at 25 ºC, except that the passenger strand, but not the guide strand, was 5ʹ 32 P-radiolabeled, and the target was RL 1x mm1 mrna or RL 1x mm5 mrna, which contained a central-bulged target site complementary to the passenger strand of duplex A or duplex B, respectively. Only complex I was detected when the passenger strand was radiolabeled. (b) Native gel analysis was performed using a series of non-radiolabeled target RNAs with different lengths. Migration of complex II, but not that of complex I, was affected by the length of the target mrna, indicating that only complex II can recognize the target. Nature Structural & Molecular Biology: doi:1.138/nsmb.163

3 Kawamata et al., Figure S3 a b c gw182[rnai] + dcr-1[rnai] + dcr-2[rnai] Flag-Ago GW182 Dcr-1 Dcr-2 Flag-Ago1 Pre-let-7 dcr-2[rnai] + Flag-Ago1 dcr-1[rnai] + RL/FL Luminescence (normalized) 1..5 Mature let-7 Control gw182 [RNAi] dcr-2[rnai] + Flag-Ago1 dcr-1 [RNAi] d e dcr-2[rnai] + Flag-Ago1 dcr-2[rnai] + Flag-Ago1 Control gw182 [RNAi] dcr-1 [RNAi] Duplex A Duplex B Time (min) Target RNA Cleaved Control gw182[rnai] dcr-1[rnai] Control gw182[rnai] dcr-1[rnai] Supplementary Figure 3 Dcr-1 and GW182 are dispensable for Ago1-RISC assembly per se. (a) Flag-tagged Ago1 was expressed in S2 cells in which either Dcr-2 alone, Dcr-2 and Dcr-1, or Dcr-2 and GW182 were depleted by RNAi, confirmed by Western blotting. (b) Processing of pre-let-7 was abolished in the dcr-1[rnai] lysate. (c) Ago1-mediated translational repression was significantly impaired in gw182[rnai] lysate. The RL/FL luminescence was normalized to the value of no RISC programming. The graph shows means and standard deviations from three independent trials. (d) Target cleavage activity of Ago1-RISC was unaffected in the absence of Dcr-1 or GW182. Duplex B was incubated with lysate for 3 min, and Flag-Ago1 was immunoprecipitated by anti-flag antibody at 4 ºC for 1 hour. The immunoprecipitated Flag-Ago1-RISC was washed three times with wash buffer containing.8 M NaCl and.1 % (v/v) triton X-1, and then target cleavage was assayed. (e) Formation of pre-ago1-risc and mature Ago1-RISC was unaffected by the depletion of Dcr-1 or GW182. Nature Structural & Molecular Biology: doi:1.138/nsmb.163 Pre-Ago1-RISC Mature Ago1-RISC

4 Kawamata et al., Figure S4 Time (min) Duplex A +ATP ATP Pre-Ago1-RISC Supplementary Figure 4 Depletion of ATP by hexokinase at 15 ºC blocks the pre-ago1-risc formation. Native gel analysis was performed using duplex A as in Figure 3a, except that endogenous ATP was depleted by.3 U µl -1 hexokinase and 2 mm glucose at 15 ºC for 3 min as in Figure 3c. The ATP regeneration system was omitted in this assay. Nature Structural & Molecular Biology: doi:1.138/nsmb.163

5 a b Kawamata et al., Figure S5 21-mer mm series (3 min cleavage) 21-mer MM1/mm series (5 min cleavage).6.2 Fraction target cleaved.4.2 Fraction target cleaved c d 25-mer mm series (4 min cleavage) 25-mer MM1/mm series (3 min cleavage).3.3 Fraction target cleaved.2.1 Fraction target cleaved.2.1 Supplementary Figure 5 Ago1-mediated target cleavage assay confirms the preference of the seed and 3ʹ-mid mismatches for Ago1-RISC maturation. (a) 21-nt mm series (mm1 mm17) were used to program Ago1-RISC at 25 ºC for 3min. Cleavage of perfectly complementary target mrna was assayed at 3 min. (b) 21-nt MM1/mm series (MM1/mm1 mm17) were used to program Ago1-RISC at 25 ºC for 3 min. Cleavage of perfectly complementary target mrna was assayed at 5 min. (c) 25-nt mm series (25 nt mm1 mm19) were used to program Ago1-RISC at 25 ºC for 3 min. Cleavage of perfectly complementary target mrna was assayed at 4 min. (d) 25-nt MM1/mm series (25 nt MM1/mm1 mm19) were used to program Ago1-RISC at 25 ºC for 3 min. Cleavage of perfectly complementary target mrna was assayed at 3 min. Note that target cleavage activity was augmented when the duplex harbored mismatches in the seed or 3ʹ-mid region, irrespective of the length of the duplex (i.e. 21-nt or 25-nt). In contrast, the cleavage activity was reduced when Nature the mismatches Structural & were Molecular at guide Biology: position doi:1.138/nsmb

6 a c Kawamata et al., Figure S6 25-nt mm series 25-nt MM1/mm series Pre-Ago1-RISC Pre-Ago1-RISC Mature Ago1-RISC b d 4 e.8 Pre-Ago1-RISC (arbitrary) Pre-Ago1-RISC (arbitrary) Mature Ago1-RISC (arbitrary) Mature/(mature + pre) Supplementary Figure 6 The position of mismatch is measured from the 5ʹ end of the guide strand, not the center of the duplex or the 3ʹ end of the guide, for both Ago1-RISC loading and maturation. (a b) 25-nt mm series (25 nt mm1-mm19) were used to assemble pre-ago1-risc at 15 ºC. (c e) 25-nt MM1/mm series (25 nt MM1/mm1-mm19) were used to monitor conversion from pre-ago1-risc to mature Ago1-RISC at 25 ºC. These native gel experiments were repeated three times with excellent reproducibility, and representative data are shown. Nature Structural & Molecular Biology: doi:1.138/nsmb.163

7 Kawamata et al., Figure S7 b MM1 Pre-Ago1-RISC UU U:G U-A 13 UU U:G U-A 5 mm5 mm9 mm15 Control (mm1) GU5 GU9 GU15 a Pre-Ago1-RISC Mature Ago1-RISC 15 ºC 25 ºC Supplementary Figure 7 G:U wobbles behave like mismatches for both Ago1-RISC loading and maturation. (a) 21-nt small RNA duplex mm series (mm1, mm5, mm9, and mm15) and GU series, where a G:U wobble was introduced at the guide position 5, 9, or 15 (GU5, GU9 and GU15), were used to assemble pre-ago1-risc at 15 ºC. mm9 and GU9 formed significantly more pre-ago1-risc than mm5, GU5, mm15 or GU15. (b) MM1 duplex derivatives containing A-U base-pair, G:U wobble, or U U mismatch at position 5 (seed region) or 13 (3ʹ-mid region) were used to assemble mature Ago1-RISC at 25 ºC. G:U Nature Structuralas&well Molecular Biology: doi:1.138/nsmb.163 wobbles, as mismatches, in the seed or 3ʹ-mid regions enhanced maturation of Ago1-RISC.

8 Kawamata et al., Figure S8 a Fly b Worm c Mouse Portion of mirnas with a mismatch in 3-nt window Guide position (center of 3-nt window) Portion of mirnas with a mismatch in 3-nt window Guide position (center of 3-nt window) Portion of mirnas with a mismatch in 3-nt window Guide position (center of 3-nt window) Supplementary Figure 8 Natural mirna/mirna* duplexes tend to have mismatches in the central region. A 3-nt window centered at each position was slid across the mirna strand (guide position 2 17), and the number of mirnas with at least one mismatch in the window were normalized by the total number of mirnas analyzed. (a) D. melanogaster (b) C. elegans (c) M. musculus. The graphs show means and standard errors. Nature Structural & Molecular Biology: doi:1.138/nsmb.163

9 Supplementary Table 1 Synthetic small RNA duplexes used in this study. 21-nt mm series of duplexes used in Figure 4a and Supplementary Figure 5a. The guide strand (red) of each small RNA duplex corresponds to the sequence of the Drosophila mirna let-7. mm1 3 -UCUCUCCAUCAUCCAACAUAU-5 mm2 3 -UCUGUCCAUCAUCCAACAUAU-5 mm3 3 -UCUCACCAUCAUCCAACAUAU-5 mm4 3 -UCUCUGCAUCAUCCAACAUAU-5 mm5 3 -UCUCUCGAUCAUCCAACAUAU-5 mm6 3 -UCUCUCCUUCAUCCAACAUAU-5 mm7 3 -UCUCUCCAACAUCCAACAUAU-5 mm8 3 -UCUCUCCAUGAUCCAACAUAU-5 mm9 3 -UCUCUCCAUCUUCCAACAUAU-5 mm1 3 -UCUCUCCAUCAACCAACAUAU-5 mm11 3 -UCUCUCCAUCAUGCAACAUAU-5 mm12 3 -UCUCUCCAUCAUCGAACAUAU-5 mm13 3 -UCUCUCCAUCAUCCUACAUAU-5 mm14 3 -UCUCUCCAUCAUCCAUCAUAU-5 mm15 3 -UCUCUCCAUCAUCCAAGAUAU-5 mm16 3 -UCUCUCCAUCAUCCAACUUAU-5 mm17 3 -UCUCUCCAUCAUCCAACAAAU-5 Nature Structural & Molecular Biology: doi:1.138/nsmb.163

10 21-nt MM1/mm series of duplexes used in Figure 4c and Supplementary Figure 5b. They are identical to the duplexes mm1-mm17 except that an additional mismatch is introduced at the guide position 1 (blue). MM1/mm1 is duplex A, and MM1/mm5 is duplex B. MM1/mm1 = duplex A MM1/mm2 MM1/mm3 MM1/mm4 MM1/mm5 = duplex B MM1/mm6 MM1/mm7 MM1/mm8 MM1/mm9 MM1/mm11 MM1/mm12 MM1/mm13 MM1/mm14 MM1/mm15 MM1/mm16 MM1/mm17 3 -UCUCUCCAUCAUCCAACAUAU-5 3 -UCUGUCCAUCAUCCAACAUAU-5 3 -UCUCACCAUCAUCCAACAUAU-5 3 -UCUCUGCAUCAUCCAACAUAU-5 3 -UCUCUCGAUCAUCCAACAUAU-5 3 -UCUCUCCUUCAUCCAACAUAU-5 3 -UCUCUCCAACAUCCAACAUAU-5 3 -UCUCUCCAUGAUCCAACAUAU-5 3 -UCUCUCCAUCUUCCAACAUAU-5 3 -UCUCUCCAUCAUGCAACAUAU-5 3 -UCUCUCCAUCAUCGAACAUAU-5 3 -UCUCUCCAUCAUCCUACAUAU-5 3 -UCUCUCCAUCAUCCAUCAUAU-5 3 -UCUCUCCAUCAUCCAAGAUAU-5 3 -UCUCUCCAUCAUCCAACUUAU-5 3 -UCUCUCCAUCAUCCAACAAAU-5 Nature Structural & Molecular Biology: doi:1.138/nsmb.163

11 25-nt mm series of duplexes used in Supplementary Figure 6a and Supplementary Figure 5c. 25-nt mm1 3 -UCUCUCCAUCAUCCAACAUAUCACU-5 25-nt mm3 3 -UCUCACCAUCAUCCAACAUAUCACU-5 25-nt mm5 3 -UCUCUCGAUCAUCCAACAUAUCACU-5 25-nt mm7 3 -UCUCUCCAACAUCCAACAUAUCACU-5 25-nt mm9 3 -UCUCUCCAUCUUCCAACAUAUCACU-5 25-nt mm11 3 -UCUCUCCAUCAUGCAACAUAUCACU-5 25-nt mm13 3 -UCUCUCCAUCAUCCUACAUAUCACU-5 25-nt mm15 3 -UCUCUCCAUCAUCCAAGAUAUCACU-5 25-nt mm17 3 -UCUCUCCAUCAUCCAACAAAUCACU-5 25-nt mm19 3 -UCUCUCCAUCAUCCAACAAAACACU-5 Nature Structural & Molecular Biology: doi:1.138/nsmb.163

12 25-nt MM1/mm series of duplexes used in Supplementary Figure 6c and Supplementary Figure 5d. They are identical to the duplexes 25-nt mm1-mm17 except that an additional mismatch is introduced at the guide position 1 (blue). 25-nt MM1/mm1 3 -UCUCUCCAUCAUCCAACAUAUCACU-5 25-nt MM1/mm3 3 -UCUCACCAUCAUCCAACAUAUCACU-5 25-nt MM1/mm5 3 -UCUCUCGAUCAUCCAACAUAUCACU-5 25-nt MM1/mm7 3 -UCUCUCCAACAUCCAACAUAUCACU-5 25-nt MM1/mm9 3 -UCUCUCCAUCUUCCAACAUAUCACU-5 25-nt MM1/mm11 3 -UCUCUCCAUCAUGCAACAUAUCACU-5 25-nt MM1/mm13 3 -UCUCUCCAUCAUCCUACAUAUCACU-5 25-nt MM1/mm15 3 -UCUCUCCAUCAUCCAAGAUAUCACU-5 25-nt MM1/mm17 3 -UCUCUCCAUCAUCCAACAAAUCACU-5 25-nt MM1/mm19 3 -UCUCUCCAUCAUCCAACAAAACACU-5 Nature Structural & Molecular Biology: doi:1.138/nsmb.163

13 21-nt small RNA duplexes used in Supplementary Figure 7. GU5 3 -UCUCUCUAUCAUCCAACAUAU-5 GU9 3 -UCUCUCCAUCGUCCAACAUAU-5 GU15 3 -UCUCUCCAUCAUCCAAUAUAU-5 5 -UGAGUUAGUUGGUUGUAUAGU-3 MM1/(U U)5 3 -UCUCUCUAUCAUCCAACAUAU-5 5 -UGAGUUAGUUGGUUGUAUAGU-3 MM1/(U:G)5 3 -UCUCUCGAUCAUCCAACAUAU-5 5 -UGAGUUAGUUGGUUGUAUAGU-3 MM1/(U-A)5 3 -UCUCUCAAUCAUCCAACAUAU-5 MM1/(U U)13 3 -UCUCUCCAUCAUCCUACAUAU-5 MM1/(U:G)13 3 -UCUCUCCAUCAUCCGACAUAU-5 MM1/(U-A)13 3 -UCUCUCCAUCAUCCAACAUAU-5 Nature Structural & Molecular Biology: doi:1.138/nsmb.163