Supplementary Information. Recognition of the pre-mirna structure by Drosophila Dicer-1

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1 Supplementary Information Recognition of the pre-mirna structure by Drosophila Dicer-1 Akihisa Tsutsumi 1,2, Tomoko Kawamata 1, Natsuko Izumi 1 Hervé Seitz 3,4 and Yukihide Tomari 1,2 Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

ΔHelicase E198A E2139A ΔDEXDc ΔHELICc pre- Catalytic mutant (E198A E2139A) 5 1 2

= WT] [S25] [S28] [S31] [S34] 5 1 2 4 5 1 2 4 5 1 2 4 5 1 2 4 5 1 2 4

distance from the 3ʹ overhang to the loop.

Approximately 2 pmol of each recombinant protein was separated by SDS-PAGE and

(b) Purified Dcr-1 catalytic mutant showed no dicing activity.

2 Tsutsumi et al. Supplementary Figure 1 a b WT (kda) Marker WT E198A E2139A ΔLinker ΔHelicase ΔHelicase E198A E2139A ΔDEXDc ΔHELICc pre- Catalytic mutant (E198A E2139A) c [L14 = WT] [L8] [L4] d [S22 = WT] [S25] [S28] [S31] [S34] Supplementary Figure 1 Dcr-1 recognizes the terminal loop and measures the distance from the 3ʹ overhang to the loop. (a) Purification of recombinant Dcr-1 and mutant proteins. Approximately 2 pmol of each recombinant protein was separated by SDS-PAGE and stained by Coomassie Brilliant Blue. (b) Purified Dcr-1 catalytic mutant showed no dicing activity. (c) Dcr-1 requires the loop region of a proper size (highlighted in blue). Quantification is shown in Fig. 1c. (d) Dcr-1 requires the stem region of a proper length (highlighted in yellow). Quantification is shown in Fig. 2b. Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

Tsutsumi et al. Supplementary Figure 2 a b 1. [Full-length Dcr-1] (nm) Dcr-1:RNA complex 22.1 22.

[ΔHelicase Dcr-1] (nm) Dcr-1:RNA complex Free RNA 4

= WT] [frayed blunt end] [paired blunt end] [Dcr-1] (nm) 22.1 22.

requirement of Dcr-1. (a) A representative gel-shift assay for full-length Dcr-1.

The average ± standard deviation is reported as K d in Table 1.

(d) Quantitative analysis of the gel-shift assays for Helicase Dcr-1 from three independent trials.

3 Tsutsumi et al. Supplementary Figure 2 a b 1. [Full-length Dcr-1] (nm) Dcr-1:RNA complex Fraction bound WT S34 L4 Free RNA [Full-length Dcr-1] (nm) c d 1. [ΔHelicase Dcr-1] (nm) Dcr-1:RNA complex Free RNA Fraction bound [ΔHelicase Dcr-1] (nm) e [paired overhang] [frayed overhang = WT] [frayed blunt end] [paired blunt end] [Dcr-1] (nm) Dcr-1:RNA complex Free RNA f Mock ( ATP) +ATP +AMP- CPP +AMP- PCP +ADP g [Dcr-1] (nm) ATP +ATP Dcr-1:RNA complex let-7 Free RNA Supplementary Figure 2 Substrate binding affinity and ATP requirement of Dcr-1. (a) A representative gel-shift assay for full-length Dcr-1. (b) Quantitative analysis of the gel-shift assays for full-length Dcr-1 from three independent trials. The average ± standard deviation is reported as K d in Table 1. (c) A representative gel-shift assay for Helicase Dcr-1. (d) Quantitative analysis of the gel-shift assays for Helicase Dcr-1 from three independent trials. The average ± standard deviation is reported as K d in Table 2. (e) A gel-shift assay with blunt or 3ʹ overhang variants with paired end or frayed end. (f) Dicing reactions were performed for wild-type and the long-stem variant [S34] in the presence of ATP or its analogues. (g) A gel-shift assay was performed in the presence or absence of ATP. Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

4 Tsutsumi et al. Supplementary Figure 3 Q HsRIG-I QNLSENSCPPSEVSDTNLYSPFKPRNYQLELALPAMKGKNTIICAPTGCGKTFVSLLICEHHLK----KFPQGQKGKVVF DmDcr MEDVEIKPRGYQLRLVDHLTK-SNGIVYLPTGSGKTFVAILVLKRFSQ--DFDKPIESGGKRAL AgDcr SQNKEP--MEDFAPRNYQVQMKEICLA-KNTIIFLPTGSGKTYIALMVMKEISH--QLRNTVHEGGKRTF CqDcr MQTIEETQKEDFVPRFYQSQMKDVCIA-KNTIVYLPTGAGKTHIALMAIRELGRRGHLEKPLSQGGKRTF CeDicer MVRVRADLQCFNPRDYQVELLDKATK-KNTIVQLGTGSGKTFIAVLLLKEYGV--QLFAPLDQGGKRAF CiDicer IELLECAAK-QNTIICLNGDFGKCFMYTMFLREVII--KHNGKLNNAPKFAV SpDicer MDEPPSSPMGLEVIDVETPGGS------SVTSQKEYTPILSFTPRHYQLELLETALE-QNTTVCLQSGTEKTFLAVMLIKELSG--SVRLRLEDGGKRTI BfDicer QREPQHLSIFTPRLYQMELLDAALE-KNTIVCLGTGSGKTFVAVMLIKELSS--QTRAALSDGGKRTF HsDicer MKSPALQPLSMAGLQLMTPASSPMGPFFGLPWQQEAIHDNIYTPRKYQVELLEAALD-HNTIVCLNTGSGKTFIAVLLTKELSY--QIRGDFSRNGKRTV DmDcr MAFHWCDNNLHTTVFTPRDFQVELLATAYE-RNTIICLGHRSSKEFIALKLLQELSR--RARRHGRVSVYLSC AgDcr MSLFHWTDGNIHTTALTPRDYQTELLATARE-ENLIVCIAHNSAKEFLAVKLIQSMRT--NRWSSHPEAPGKAI CqDcr MAHSWTD-AIHTTSLTPRDFQVELLASARE-RNLILCLAHNSNKEFIALKLIHELGF--QLRSTAQRRK-RTL Ia DEXDc Ib II HsRIG-I FANQIPVYE-QQKSVFSKYFERHGYRVTGISGATAENVP VEQIVENNDIIILTPQILVNNLKKGTIPSLSIFTLMIFDECHNTSKQHPYNMIM DmDcr-2 FMCNTVEL---ARQQAMAVRRCTNFKVGFYVGEQGVDDW---TRGMWSDEIKKNQVLVGTAQVFLDMVTQTYVA-LSSLSVVIIDECHHGTGHHPFREFM AgDcr-2 FLANTVAL---AKQQAQFFARHMPFNVRLYTSEVNVDAW---KSDRWHEEFSEGQVIICTAQILLDVLRHGYMS-PANINLIVFDECHRAVGQHPMHAIM CqDcr-2 FIVNTVAL---AKQQAEMIGRNVVFKTSVYTSDRDVDTW---KQDRWLEEFAKYQIIVCTCQILLDVLKHGYLA-MSHINLLVFDECHHGVGDHPMHGIM CeDicer FVVEKVNL---VEQQAIHIEVHTSFKVGQVHGQTSSGLWD--SKEQCDQFMKRHHVVVITAQCLLDLIRHAYLK-IEDMCVLIFDECHHALGSQHPYRSI CiDicer LLLKEDCM---VNEQSELLRSHLDVDIISCTHN-MTDSF---TDESLQQLCHDHQVLVCTPEVFVKLLSISLYI-AQYMDLVIFDDCHLVNIEMHPIRKI SpDicer FLVNSESY---LSEYGNVIHTHTDLNVKEYRNSDKMISW---DRTRWQDEIEHCHVAVMDSEIFLKLLQMSYVG-LSMLNLLILDECHHTLQHLHPYHQI BfDicer FLVNNVPL---VSQQAAVITTHTNLSIGEYVGAMGVDLW---TRDRWQQEFDTHHVLVMTAQIFLDILQHGFLP-LSKVNLLIIDECHHAVGH-HPYREI HsDicer FLVNSANQ---VAQQVSAVRTHSDLKVGEYSNLEVNASW---TKERWNQEFTKHQVLIMTCYVALNVLKNGYLS-LSDINLLVFDECHLAILD-HPYREI DmDcr-1 EVGTSTEPCS-IYTMLTHLTDLRVWQEQP------DMQIP------FDHCWTDYHVSILRPEGFLYLLETRELL-LSSVELIVLEDCHDSAVYQRIRPLF AgDcr-1 YL-TRMDRSL-LSSMVSNLTDLQVANVDDVEDSEGSHEPDGASNTPVTDVASADVLFFGSETTLLQYIEQGTVR-VQDISLLIVDECHKNYGRQELWEIC CqDcr-1 YI-SNNDS---VYSLMRNLTDLKVVHVD GDEPD------WERIVADYQVIIANERKCLDAVVCGYLE-LDEVNLLVIDECHKIYGNVEIAELF III HsRIG-I FNYLDQKLGGS-SGPLPQVIGLTASVGVGDAKNTDEALDYICKLCASLDASVIATVK-HNLEELEQVVYKPQKFFRKVESRISD-----KFKYIIAQLMR DmDcr-2 RLFTIANQT-----KLPRVVGLTGVLIKGNE--ITNVATKLKELEITYRGNIITVSDTKEMENVMLYATKPTEVMVSFP-HQEQVL---TVTRLISAEIE AgDcr-2 KEIVAAPAS-----ERPRVLGLSGTLLFKELKMASQVPDELERLENTFSSTIATVANYDDYATVASFSTNPNEVLVTYSKPAVHLM---PLVKEMWPRID CqDcr-2 EQYLRARKE-----DRPRVIGLSGMLLYKELKMKEQVAQELERLENTFDSTIATVGSYDAYTEVCRFSTDPKEGLLSFH--VVQSS---DVMRNLQGQIN CeDicer MVDYKLLKK---DKPVPRVLGLTASLIKAKVAP-EKLMEQLKKLESAMDSVIETAS---DLVSLSKYGAKPYEVVIICKDFEIG-CLGIPNFDTVIEIFD CiDicer MKILDENR------YTPMILGLTSSLLNNNITA-DELNKCIHSMETVMHCKAQTAT---DLIAVDRHNINPPVENIILSSDENKDGLNSQLLSDIRKILN SpDicer MLAFKQYPR----DDRPRILGLTESILKGDFKP-CALEEHIDMLETSLQSKAKIAT---NLTSISKYGTKPKEAVIVCRKYEDC----IELVSKLSKLLT BfDicer MKTFDTCQV----QDYPRVLGLTASILQGKCAP-DSLFQRVRNLEVTLRSSAETAT---DLVGVDRYTTQPNEVVIESGPAQNE----PGLSQALQTLVD HsDicer MKLCENCP------SCPRILGLTASILNGKCDP-EELEEKIQKLEKILKSNAETAT---DLVVLDRYTSQPCEIVVDCGPFTDR----SGLYERLLMELE DmDcr-1 ENHIMPAP----PADRPRILGLAGPLHSAGCEL-QQLSAMLATLEQSVLCQIETAS---DIVTVLRYCSRPHEYIVQCAPFEMD-----ELSLVLADVLN AgDcr-1 ARLTHQAPSSDRPAQRTRILGLAGPLHGAGCTP-ERLCWELHYLERCLRARIETAS---DITSVLRFSTKPTELILECIPPKPS-----NLTQLLRMLIQ CqDcr-1 SEYYERCR------ERPKILGLAGPLHNAGCIP-GRLSAELEQLERCLKAKAETAS---DIVTVLRYCTKPKELILQCAPPQQS-----ELALYLREIVE HsRIG-I DTESLAKRICKDLENLS QIQNREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKY DmDcr-2 KFYVSLDLMNIGVQPIRR SKSLQCLRDPSKKSFVKQLFNDFLYQMKEYGIYAASIAIISLIVEFDIKRRQAET-LS AgDcr-2 AFVEWLLQVYLPEYSTQS TRTLQKSFCKPLK-EVKRALTEFKHQLEEHGMYAGSLAILAVIVQLEVSKRQSPC-DK CqDcr-2 DFINKVALFDLPKLLNQ NKSLMRDMPKPKK-VITKYFKELNYQFEDLGLFGGAIALLGLIVQFELDKRESDH-TM CeDicer ETVAFVNTTTEFHPDLD LDPRRPIKDSLKTTRAVFRQLGPWAAWRTAQVWEKELGKIIKSQVLPDK CiDicer GALEWLNSCQKEKQEED LDVCTVAIQLINESLMVADVLGPWCVYQLIVVVLKQLQKNLHLINW-MW SpDicer DGLDQLNQFEYSFKARDFEG ELVDCKRDPVLPARQALMECLSTLRTIGPLGVKILIPMLVREILKLEKHEFL-DV BfDicer DALAFISSCKVRVSVEEGE RDPCQVPKQAIQECRAILEVLGTWATRRAAILINRELEKLIKHEWS-EQ HsDicer EALNFINDCNISVHSKE RDSTLISKQILSDCRAVLVVLGPWCADKVAGMMVRELQKYIKHEQE-EL DmDcr-1 THKSFLLDHRYDPYEIYG TDQFMDELKDIPDPKVDPLNVINSLLVVLHEMGPWCTQRAAHHFYQCNEKLKVKTPH-ER AgDcr-1 RQIAFLKQHRYEPLAVYGLDGNDSASDKPDTDGEPEKDEENDDLRRELKSIPDPTVGPLSYLKQYLELLDEFGPWGADRGALELLTTIDQEKVKVPY-DR CqDcr-1 TQMTFLQEHRYDPSEIY EDDEFLEELKHIPDPKVDPLNFLREFLTVLDEMGPWCADRAALALIVQIEKRKIKTPY-ER I HsRIG-I NDALIISEHARMKDALDYLKDFFSNVRAAG FDEIEQDLTQRFEEKLQELESVSRDPSNENPKLEDLCFILQEEYHLN DmDcr-2 VKLMHRTALTLCEKIRHLLVQKLQDMTYDDDDDNVNTEEVIMNFSTPKVQRFLMSLKVSFADKD AgDcr-2 ARQVYRSAISFCESIRHELVEAMSGLRGTHQ ILSFSSDQARKLLKYLEDSYRTAE CqDcr-2 LRLLYRSCITFCENLRHQLENVMQGLEIKKK LTLFSSIKARQLIAQLEGFYKEGR CeDicer TLRFLNMAKTSMITIKRLLEPEMKKIKS IEALRPYVPQRVIRLFEILETFNPEFQKERMKLEK CiDicer QAVMLQYVQTKFKMVYSICHWYMG-DQKA VNLAFVAPKVHRLIQVLKEYKPVVEGEEKQNTSFKYNNRN ESDYVSWWQD-- SpDicer HLLFLQHATTQLRMVERICDNALREGGNF YDLKFLTPKVRRLLEVLSGFKPKEV--SPEEKAQQSTNRE RANKFASRRK-AH BfDicer HQLFLRWVHTVMHHVHLLCDEAFK-DVED VDVQFVTPKVRRLLEVLRECKPNEV---QSDNSKSVSNMNS KNHYHSSRR--- HsDicer HRKFLLFTDTFLRKIHALCEEHFS-PAS LDLKFVTPKVIKLLEILRKYKPYER--QQFESVEWYNNRN QDNYVSWSD--- DmDcr-1 HYLLYCLVSTALIQLYSLCEHAFHRHLGSGS----DSRQTIERYSSPKVRRLLQTLRCFKPEE--VHTQADGLRRMRHQVDQADFNRLSHTLESKCRMVD AgDcr-1 HFLLFCMVYTTLLQARATVASVFAQHD TELERIKRYSTPKVRRLLEVLAWFGEQR--NRPKDRNQPATLHHHQQVHNQ--QRILYCFCRNVE CqDcr-1 HFLLLCLVSTVFVQIRSYCDLVFQQYA TELERIEAHSTPKVLRMLEIFRLFRPDG-ANRGTVSSKAPIKEVTESCQ----LPSCQSLLKEIK Linker-1 HsRIG-I DmDcr AgDcr CqDcr CeDicer CiDicer SDAEDEDADMMEGKKEEKEDENHP SL SpDicer RSHKDIKNPRWRP-IVPEDQDEDEVSDEEEEEEEDEEDVGLQQ TGR BfDicer RDNLDNQD PNTSFQ HsDicer SEDDDEDEEIEEKEKPETNFPSP DmDcr-1 QMD QPPTETRALVATLEQILHTTEDRQT---NRSAARVTPTPTPAHAKP KPSSGANTAQPRTRRRVYTRR-HHRDHN---D AgDcr-1 CKELE KSYHTFGAQIADVDERIKQLDGLLASVRKRTERLNLKHKVVDNAAAEGGGTLGVQSPRHGHESRANNFRRKRFAGGGHSHHRSN--D CqDcr-1 TVDFQ KFVDRVETVTSSIAIITEGLESLKTSVKSISDAPPLKQNLVFNEKK PRSPFKNGIRQIRQRKKIGFTHRNRSNFHNQ--N IV HELICc IVa V Va HsRIG-I --PETITILFVKTRALVDALKNWIEGNPK LSFLKPGILTGRGK TNQNTGMTLPAQKCILDAFKASGDHNILIATSVADEGI DmDcr-2 -PKDICCLVFVERRYTCKCIYGLLLNYIQ STPELRNVLTPQFMVGRN-----NISPDFESVLERKWQKSAIQQFRDG-NANLMICSSVLEEGI AgDcr-2 -DKNKQALVFVKRRFTAKVLYHLIRIYFHCLSKRDNEDELVEPIVKPDFIVGANA----ALEESIDAILVVREDRRVIENFRKR-KINVLCATNVLEEGI CqDcr-2 -SRKTKTLIFVQRRFSAKVVYHLLKIYFS-----ETEN---AETILPDFMVGCNG----TMPESIEQILSAKKDRRVLERFKKN-ETNVIVTTNVLEEGI CeDicer -AEHLSAIIFVDQRYIAYSLLLMMRHIKS WEPKFKFVNPDYVVGASG----RNLASSDSQGLHKRQTEVLRRFHRN-EINCLIATSVLEEGV CiDicer FINTLCGIVFVEERYVAMMLQRLLRELSK EDKNLKHVSSSHLTPGVE---GERRGVDPASMDQSKQEEVLRKFRSH-ESNLLICTGDLEDCA SpDicer STTNLCGIIFVEQRYTAVVMSRLFKKLSK RDPNLHFLLCSCIAMGSGRLGTTKKSAIQVQNQRRKHEEVLRKFRRR-EVNVLVATSSVEDGV BfDicer ----LCGIVFVERRYTAVVLNKLLQEFAK SDPDLAYISSSCIT-GHG-LSSRGMRSRETEMAFRRQEEILRRFRMH-ENNLLIGTSVVEEGV HsDicer FTNILCGIIFVERRYTAVVLNRLIKEAGK QDPELAYISSNFIT-GHG-IGKNQPRNKQMEAEFRKQEEVLRKFRAH-ETNLLIATSIVEEGV DmDcr-1 GSDTLCALIYCNQNHTARVLFELLAEISR RDPDLKFLRCQYTTDRVAD---PTTEPKEAELEHRRQEEVLKRFRMH-DCNVLIGTSVLEEGI AgDcr-1 TTDALCGLIFCNNRAMARILYVLLYEVSR SQREFEFISPQYTVDKVATN--PQNCLKQTTIEHRKQEEVLKRFRMH-ECNLLIGTSVLEEGI CqDcr-1 DPDALCALIFCNSKFTTKILYSLLYEAVR SDPQLSYINVQYTVDKSAN---PITEPKEAEVEHRKQEEVLKRFRMH-ECNLLIGTSVLEEGI Va Vb VI Linker-2 HsRIG-I DIAQCN---LVILYEYVGNVIKMIQTRGRGRARGSKCFLLTSNAGVIEKEQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEK DmDcr-2 DVQACN---HVFILDPVKTFNMYVQSKGRARTTEAKFVLFTAD AgDcr-2 DLQMCN---MVIMYDAPLSYASFMQSKGRARMKTSTYLMMTPA CqDcr-2 DLQMCN---SVIKFDYPETFASYEQSKGRARMKDSTYTVMLDS CeDicer DVKQCN---LVIKFDRPLDMRSYVQSKGRARRAGSRYVITVEE CiDicer YMPKCN---LIMRFDVPKSYRSYAESKSRARAQTSAYVMLVRE SpDicer ELPRCNGCNLVVRFDRPTSYQSYMQSKAKARAPTSHYLMLIYE BfDicer DVPKCN---LVVRFDLPKDYRSYVQSKGRARAQGSHYVMLVQQ HsDicer DIPKCN---LVVRFDLPTEYRSYVQSKGRARAPISNYIMLADT DmDcr-1 DVPKCN---LVVRWDPPTTYRSYVQCKGRARAAPAYHVILVAPSYKSPTVGSVQLTDRSHRYICATGDTTEADSDSDDSAMPNS--- AgDcr-1 ELPKCN---LVIRWNSPANYRSYAQCKGRAKAPGAYHVLFVTP--ENAASRNEQQEDMAS CqDcr-1 DLPKCN---LVIRWNEPVSYRSYVQCKGRARAPSAYHILIVTPKVDQLLAANGARDDLSEQVHRKVCER Supplementary Figure 3 Multiple alignment of the amino acid sequences of representative Dicer proteins. ClustalX 1 with default parameters was used for the alignment. The conserved motifs (motif I VI) were annotated according to refs. 2 and 3 and are shown in thick black lines. DEXDc and HELICc domains are shown in green and red lines, respectively. The two linkers specific for insect Dcr-1 proteins are shown in gray lines. Hs, Homo sapiens; Dm, D. melanogaster; Ag, Anopheles gambiae; Cq, Culex quinquefasciatus; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Sp, Strongylocentrotus purpuratus; Bf, Branchiostoma floridae. Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

gambiae Dcr-1 (Mosquito) C. elegans Dicer (Worm) C.

5 Dcr-1 proteins specific for pre-mirnas H.

for long dsrnas Supplementary Figure 4 A phylogenic tree of

5 Tsutsumi et al. Supplementary Figure 4 Single but dual functional Dicer proteins D. melanogaster Dcr-1 (Fly) H. sapiens Dicer (Human) B. floridae Dicer (Lancelet) S. purpuratus Dicer (Sea Urchin) C. intestinalis Dicer (Sea Squirt) A. gambiae Dcr-1 (Mosquito) C. elegans Dicer (Worm) C. quinquefasciatus Dcr-1 (Mosquito).5 Dcr-1 proteins specific for pre-mirnas H. sapiens RIG-I (Human) D. melanogaster Dcr-2 (Fly) A. gambiae Dcr-2 (Mosquito) C. quinquefasciatus Dcr-2 (Mosquito) Dcr-2 proteins specific for long dsrnas Supplementary Figure 4 A phylogenic tree of representative Dicer proteins. The multiple sequence alignment in Supplementary Fig. 3 was used to draw the tree. The scale bar indicates the branch length for a substitution frequency of.5 per amino acid. Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

5 124 5 124 ΔHelicase ΔDEXDc ΔHELICc Supplementary Figure 5 Dcr-1 binds the terminal loop via its helicase domain.

6 Tsutsumi et al. Supplementary Figure 5 a b WT ΔLinker c d e ΔHelicase ΔDEXDc ΔHELICc Supplementary Figure 5 Dcr-1 binds the terminal loop via its helicase domain. (a e) Dicing efficiency for and its structural derivatives by wild-type (a), Linker (b), Helicase (c), DEXDc (d), and HELICc (e) Dcr-1 proteins. Quantification is shown in Figs. 3c g. Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

7 Tsutsumi et al. Supplementary Figure 6 a Fraction diced dcr-1[rnai] d Fraction diced Dcr-1 only b Fraction diced dcr-1[rnai] +WT Dcr-1 rescue e Fraction diced Dcr-1/Loqs-PB c 1..8 dcr-1[rnai] +ΔHelicase Dcr-1 rescue Fraction diced Supplementary Figure 6 Neither N-terminal SBP-tag nor Loqs affects Dcr-1 s substrate specificity. (a) Dicing assays of wild-type of and the long-stem and small-loop mutants were performed in lysate from S2 cells where endogenous Dcr-1 was knocked down by RNAi. (b) Same as in a but in lysate from S2 cells where endogenous Dcr-1 was knocked down by RNAi and then no-tagged intact Dcr-1 was complemented. (c) Same as in a but in lysate from S2 cells where endogenous Dcr-1 was knocked down by RNAi and then no-tagged Dcr-1 Helicase mutant was complemented. (d,e) Dicing assays for and its structural derivatives by Dcr-1 alone (d) or Dcr-1 Loqs-PB complex (e). Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

Fly Dicer-1 D. melanogaster C. elegans H. sapiens M. musculus D. melanogaster C. elegans H. sapiens M. musculus b e 6 Loop size let-7 duplex [S22;ss7+7] 5 1 2 4 5 1 2 4 5 1 2 4 5 1 2 4 5 4 Length (nt) 3 2 1 D.

8 Tsutsumi et al. Supplementary Figure 7 a let-7 duplex [S22;ss7+7] c 8 Distance from the 5ʹ end to the loop d 8 Distance from the 3ʹ end to the loop Length (nt) 4 Length (nt) Fly Dicer-1 D. melanogaster C. elegans H. sapiens M. musculus D. melanogaster C. elegans H. sapiens M. musculus b e 6 Loop size let-7 duplex [S22;ss7+7] Length (nt) D. melanogaster C. elegans H. sapiens M. musculus Human Dicer Supplementary Figure 7 Comparison of Dicer s substrate specificity and pre-mirna structure among species. (a,b) Unlike fly Dcr-1, human DICER1 shows loose substrate specificity. Dicing assays for and its structural derivatives by fly Dcr-1 (a) or human DICER1 (b). (c e) Statistical analysis of the structural heterogeneity of 123 pre-mirnas in D. melanogaster, 134 pre-mirnas in C. elegans, 42 pre-mirnas in H. sapiens and 362 pre-mirnas in M. musculus: Distance from the 5ʹ end to the loop (c), distance from the 3ʹ end to the loop (d), and loop size (e). Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

9 Supplementary Table 1. The sequences of RNA substrates used in this study. Name let-7 shrna let-7 shrna [mm9] [L8] [L4] [paired overhang] [frayed blunt end] [paired blunt end] [S25] [S28] [S31] [S34] let-7 duplex [ss7+7] Sequence A C 3ʹ -UCU G G ACUACA 5ʹ - pu GUAAUU GAGGUAGUAGGUUGUAUAGUA A CUCCAUCAUCCAACAUAUCAU C 3ʹ -UCU ACUACA 5ʹ - pu U GUAAUU GAGGUAG AGGUUGUAUAGUA A CUCCAUC UCCAACAUAUCAU C 3ʹ -UCU U ACUACA 5ʹ - pu G G GUA A C 3ʹ -UCU G G ACU 5ʹ - pu G G G A C 3ʹ -UCU G G A G G GUAAUU 5ʹ - pu A A C 3ʹ -UC G G ACUACA A C 3ʹ -U G G ACUACA G G GUAAUU 5ʹ - pu A 3ʹ -A C G G ACUACA GAA A CUU C 3ʹ -UCU G G ACUACA GAACAU A CUUGUA C 3ʹ -UCU G G ACUACA GAACAUUCG A CUUGUAAGC C 3ʹ -UCU G G ACUACA GAACAUUCGUCA A CUUGUAAGCAGU C 3ʹ -UCU G G ACUACA A-3ʹ 3ʹ -UCU G G ACUACAC-5ʹ 1 Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

10 let-7 duplex [ss7+3] let-7 duplex [ss3+7] let-7 duplex [ss3+3] let-7 duplex [ss15+3] let-7 duplex [ss3+15] let-7 duplex [S34;ss7+7] let-7 duplex [S22;ss7+7;S12] pre-mir-8 pre-mir-8 [L4] pre-mir-8 [S34] 5ʹ - pu G G GUAUUA-3ʹ 3ʹ -UCU G G ACU-5ʹ 5ʹ - pu G G GUA-3ʹ 3ʹ -UCU G G ACUACAC-5ʹ 5ʹ - pu G G GUA-3ʹ 3ʹ -UCU G G ACU-5ʹ 5ʹ - pu G G GAACAUUCGUAUUA-3ʹ 3ʹ -UCU G G ACU-5ʹ 5ʹ - pu G G GUA-3ʹ 3ʹ -UCU G G CUUGUAAGACUACAC-5ʹ A-3ʹ GAACAUUCGUCA CUUGUAAGCAGU 3ʹ -UCU G G ACUACAC-5ʹ 5ʹ - pu G G GAACAUU CGUCAGUAAUUA-3ʹ GCAGUCAUUAAU-5ʹ 3ʹ -UCU G G GCAUACC - G C UCCUUU 5ʹ - pcaucuu ACC GGCAG AUUAGA U UGUAGAA UGG CUGUC UAAUCU U 3ʹ -C A A A CAAUA - G C U 5ʹ - pcaucuu ACC GGCAG AUUAGA G UGUAGAA UGG CUGUC UAAUCU A 3ʹ -C A A A C - G C UCCUUU 5ʹ - pcaucuu ACC GGCAG AUUAGACUCAGCACAUGG U UGUAGAA UGG CUGUC UAAUCUGAGUCGUGUACC U 3ʹ -C A A A CAAUA 2 Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

11 Supplementary References 1. Larkin, M. A. et al. Clustal W and Clustal X version 2.. Bioinformatics 23, (27). 2. Fairman-Williams, M. E., Guenther, U. P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr Opin Struct Biol 2, (21). 3. Jankowsky, E. RNA helicases at work: binding and rearranging. Trends Biochem Sci 36, (211). 3 Nature Structural & Molecular Biology: doi:1.138/nsmb.2125

Structure and RNA-binding properties. of the Not1 Not2 Not5 module of the yeast Ccr4 Not complex

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