doi: 10.1038/nature05841 SUPPLEMENTARY INFORMATION www.nature.com/nature 1
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a Hours of Development: eif-6(rnai): 4 wildtype 30 4 lin-4 (e912lf) 30 LIN-14 LIN-28 EIF-6 b 140 TUBULIN ACTIN 1 2 3 4 5 6 7 8 9 101112 Wildtype (N2) 1 100 % Max 80 60 LIN-14 LIN-28 (top) LIN-28 (bottom) ACTIN 40 c 0 180 4hr vector(rnai) 4hr eif-6(rnai) hr vector(rnai) hr eif-6(rnai) 30hr vector(rnai) 30hr eif-6(rnai) lin-4(e912lf) 160 140 1 % Max 100 80 LIN-14 LIN-28 (top) LIN-28 (bottom) ACTIN 60 40 0 4hr vector(rnai) 4hr eif-6(rnai) hr vector(rnai) hr eif-6(rnai) 30hr vector(rnai) 30hr eif-6(rnai) SUPPLEMENTARY FIGURE 5 www.nature.com/nature 5
Hours of Development: 4 30 eif-6(rnai): - + - + - + pre-rrna precursors 18S rrna 5.8S rrna 1 2 3 4 5 6 SUPPLEMENTARY FIGURE 6 www.nature.com/nature 6
Band Name Name of Protein Locus # of sequences TRBP-AP1 Endoribonuclease Dicer Q9UPY3 40 TRBP-AP2 KIAA1631 (Mov 10) BAB13457 17 TRBP-AP3 RPL7a AAI05291 22 TRBP-AP4 RPL7 CAA41027 27 RPL13 AAH04954 10 RPL10a AAH41308 6 TRBP-AP5 RPL10a AAH41308 18 RPL14 AAH71913 12 RPL13 AAH04954 11 RPL19 Q3T0W9 8 p27bbp (eif6) AAK39426 9 RPL15 AAG44837 3 TRBP-AP6 RPL13 AAH04954 9 RPL15 AAG44837 16 RPL18 NP_000971 8 RPL19 AAL29467 3 TRBP-AP7 RPL18 AAH719 11 RPL21 XP_940793 7 RPL24-like AAH08499 4 RPL11 AAD460 3 similar to RPL29 XP_210334 3 TRBP-AP8 RPL12 NP_000967 15 RPL32 XP_345558 5 RPL28 NP_000982 4 TRBP-AP9 RPLP1 CAG47005 3 RPL27 1909362A 7 similar to RPL32 XP_345558 8 RPL12 NP_000967 6 RPLP2 NP_777213 5 RPL28 NP_000982 8 RPL34 AAC41916 5 RPL36 XP_219699 3 TRBP-AP10 RPL30-prov AAH53758 19 RPL36 XP_219699 8 RPLP2 NP_777213 7 RPL34 AAC41916 10 RPL37A AAH00555 5 RPL35A NP_067087 5 Supplementary Table 1. www.nature.com/nature 7
Supplementary Figure 1. a, Colloidal blue staining of the large TRBP-containing complex. The excised bands are delineated on the left. Contaminating bands (SKB1, MEP50, alpha-tubulin) as well as Dicer break-down products are shown using asterisks. TRBP-AP denotes TRBPassociated proteins. b, Western blot analysis of Flag-TRBP affinity eluate from H1299 cell lines using antibodies to the right of the figure. Supplementary Figure 2. a, Western blot analysis of Flag-Dicer affinity eluate isolated following increasing concentration of KCl salt washes. b, Western blot analysis of Flag-eIF6 affinity-eluate using antibodies shown to the left of the figure. Asterisk denotes a non-specific band, which is the consequence of antibody leaching from the affinity matrix c, Western blot analysis using anti-flag antibodies for the expression of Flag-tagged proteins shown on the top of the figure. d, Northern blot analysis of whole cell lysate (input) or Flag-affinity eluate (IP) using probes to mirna 23b. e, Knock-down of eif6 results in decreased eif6 RNA (right) and protein (left) levels. Supplementary Figure 3. Knock-down of eif6 or Armitage disrupts let-7b-mediated silencing. a, Transfection of a second sirna against eif6 in Hela cells results in depletion of eif6 mrna levels. b, Disruption of let-7b silencing of the reporter plasmid containing 2 let-7b binding sites following depletion of eif6. c, Knock-down of human Armitage using two different sirnas results in disruption of let-7b silencing. Supplementary Figure 4. mir-125b-mediated translational repression is diminished by eif6 knock-down. a, A diagrammatic representation of Lin-28 3 UTR displaying mir-125b binding sites. b, Depletion of eif6 rescues mir-125b mediated translational repression. Dual luciferase reporters (Firefly Luciferase contains the mirna binding sites and Renilla was used as a www.nature.com/nature 8
transfection control) were co-transfected with a mature mir-125b duplex. Each point represents at least three independent experiments. c, Depletion of BOP1 decreases the expression of the mir-125b-responsive as well as the control reporter. Supplementary Figure 5. a, Protein samples from a second, independent RNAi experiment conducted with wildtype and lin-4(e912lf) worms were collected and analyzed as in Figure 4b. b, Graphical representation of average protein levels from two independent RNAi experiment trials in wildtype and lin-4(e912lf) worms. LIN-14, both isoforms of LIN-28, and ACTIN levels were normalized to TUBULIN. Values for the vector(rnai) control at the 4 hour timepoint were set at 100% and all other samples were measured against this. Error bars correspond to +/- one standard deviation. Supplementary Figure 5. The Northern blot shown in Figure 4a was re-probed to detect the indicated precursor and mature rrnas. Supplementary Table 1. Mass spectrometric identification of TRBP-associated proteins. identity and number of polypeptides recovered in each band is shown. The www.nature.com/nature 9