Supplemental Data Antisense-Mediated Depletion Reveals Essential and Specific Functions of MicroRNAs in Drosophila Development

Supplemental Data

Antisense-Mediated Depletion

Reveals Essential and Specific Functions

of MicroRNAs in Drosophila Development Dan Leaman, Po Yu Chen, John Fak, Abdullah Yalcin, Michael Pearce, Ulrich Unnerstall, Debora S. Marks, Chris Sander, Thomas Tuschl, and Ulrike Gaul Figure S1. Northern Blots for All Drosophila miRNAs Figure S1

Note that since miRNAs differing in only a few bases are expected to crosshybridize, only one miRNA per multicopy group was tested.

Figure S2. Sequence Alignments for the Six Predicted miR-2 Family Target Sites in the 3′UTRs of the Proapoptotic Factors hid, grim, rpr, and skl. rpr

rpr201_vir ----CCCCUAAGUUUACUCAUCAAAGCGAUUGUGAUAAUGGUUUUGUUUCUUUCGAUAU

rpr201_pse UUUUUUUUUUAGUUUAAUCAUCAAAGCGAUUGUGAUAAUGGUUUUGUUUCUA-CGAAAA rpr201_ana CAAAAUUUUUAGUUUACUCAUCAAAGCGAUUGUGAUAAUGGUUUUGUUUCUA-AAAAAA rpr201_yak ACCCACUUUUAGUUUACUCAUCAAAGCGAUUGUGAUAAUGGUUUUGUUUCUA-CGAAAA rpr201_mel ACCCAAUUUUAGUUUACUCAUCAAAGCGAUUGUGAUAAUGGUUUUGUUUCUA-CAAAAA

987654321 mir-2a GAGUAGUUUCG GACACUAU -29.5 C ACC -------------------------------------------------------------------------------------------- miR-13a UGAGUAGUUUU GACACUAU -24.3 ACC -------------------------------------------------------------------------------------------- miR-11 UCG UGAGU CUGACACUA -18.7 UUCU C -------------------------------------------------------------------------------------------- miR-6 UCG UGACACUAU -16.9 UUUUUCUUG G -------------------------------------------------------------------------------------------- miR-308 GAGUG GACACUA -13.9 UCAUAUUAG A -------------- ---------------------------------------------------------------------------- grim

grim85_vir --UUUUGG--UGGCAG-CUCAAUCAAAGCGCAUUGUGAUUUUUUGGGAUUAUGGAGUUU- grim85_moj UUUUUUGG--UGGCAG-CUCAAUCAAAGCGCAUUGUGAUUUUUCCG-AGGAUGGAGAC-- grim85_pse --CGGUGG--CGGCAGGCUCAAUCAAAGCGCAUUGUGAUUUUUUUUUUUUUUUGGGAAUU grim85_ana --UCAUGGAAAGGCAGGCUCAAUCAAAGCGCAUUGUGAUUUCUUUUU------GGGUAU- grim85_yak --UCUGGGAAAGGCAGGCUCAAUCAAAGCGCAUUGUGAUUUCUUUUUU-----GGGUAU- grim85_mel --UCUGGGAAAGGCAGGCUCAAUCAAAGCGCAUUGUGAUUUCUUUUUU-----GGGUAU-

987654321 mir-2a GAG UAGUUUCG GACACUA -27.5 C ACC U --------------------------------------------------------------------------------------------- miR-13a UGAG UAGUUUU CG ACACUA -20.1 AC U --------------------------------------------------------------------------------------------- miR-6 UUCUU UCG GUGACACUA -18.8 UUU G U --------------------------------------------------------------------------------------------- miR-11 UCGUUC AGU UGACACUA -17.6 UUG C C --------------------------------------------------------------------------------------------- miR-308 UGUC UUAG GACACUAA -14.3 GAG AUA ---------------------------------------------------------------------------------------------

hid

hid778 _vir AGAAGAACAUUUUUGUAUAUAGUUUAUA---UGCCAUUUUUGUGAUGAUUUU-GUCUAU hid778 _moj AUGU-----UUUUUGUAUAUAGUUGAUAUAAUGCCAUUUUUGUGAUGAUUUUUGUCUAU hid778 _pse GUUU-UUUUUAUUUGUAUAUAGUUUUUA---UGCCAUUUUUGUGAUGUUUUGUGUCUAC hid778 _ana AUCA-UUUAAUUUUGUAUAUAGUUUUUA---UGCCAUUUUUGUGAUGUUUUGUGUCUAC hid778 _yak UUAA-AUUAAUUUUGUAUAUAGUUUUUA---UGCCAUUUUUGUGAUGUUUUGUGUCUAC hid778 _mel UUAA-AUUAAUUUUGUAUAUAGUUUUUA---UGCCAUUUUUGUGAUGUUUUGUGUCUAC

987654321 miR-6 CGGUG ACACUAU -16.0 UUUUUCUUGU --------------------------------------------------------------------------------------------- miR-308 GUGUCA AU AGGACACUA -15.6 GA U U A --------------------------------------------------------------------------------------------- miR-11 UCG UG GU GACACUAC -13.5 UUC A CU --------------------------------------------------------------------------------------------- mir-2c GGUAG GACACUAU -13.2 CG UUUCGACC --------------------------------------------------------------------------------------------- miR-13b CAG U AC GACACUAU -11.8 UGAG UUU C --------------------------------------------------------------------------------------------- hid468_vir AUAUGUAAAAUUCAAGUAAAACUGUAAACUGCCAUCUUUUGAGAUACUUUUUGUAG------- hid468_moj -------------AAUCAACUA--CAAACUGCCAUUU-UUGAGAUACUUUUUGAGAUACAUUU hid468_pse -----CAACAAAAGAACAAAAG-GAAAACUGCCAAUUCUUGAGAUAUAGUAC----------- hid468_ana AAAAAAAAUAUAUAAAGCAAAA-GAAAACUGCCAACUUUUGAGAUACUUUUGAUAA------- hid468_yak ----GAGAGAGAUAAGCAAAAAAGAAAACUGCCACUU-UUGAGAUACUUUUGAUAA------- hid468_mel ----GAGAGAGAUAAGCAAAA--GAAAACUGCCACUU-UUGAGAUACUUUUGAUAA-------

987654321 miR-6 UUUU CUU CGGUG AC CUAU -19.4 U GU A ---------------------------------------------------------------------------------------------

skl

skl57_vir A----ACACGAGGC----UGAUGGCUCCUAA--GCGCAACUUGUGAUUACUUUUC-GUUGG---- skl57_moj ACUCAAAACGAGGCC-UCUGAUGGCUCCUAA--GCGCAACUUGUGAUUACUUUUC-GUUGG---- skl57_pse ACCGUGCCCGGAGCUCUGAAGCGGCUCCUAAAAGCGCAACUUGUGAUUACUUUACCGUUGGCGAA skl57_ana G----AGGCGCCAAC-U---AAAGCUCCUAAAAGCGCAACUUGUGAUUACUUUAC-GUUGG---- skl57_yak AAAUUAAGCACCAAC-U---AAGGCUCCUAAAAGCGCAACUUGUGAUUACUUUAC-GUUGG---- skl57_mel AACUUAAGCACCAAC-U---AAGGCUCCUAAAAGCGCAACUUGUGAUUACUUUAC-GUUGG----

987654321 mir-2b CGAGGA UUUCG GACACUA -26.6 G ACC U --------------------------------------------------------------------------------------------- miR-13b UGAG GUUUU CG ACACUA -15.4 CA AC U --------------------------------------------------------------------------------------------- miR-11 UCGU UUG A UCUG ACACUA -17.3 UC G C --------------------------------------------------------------------------------------------- miR-6 UUUUC UGU GG ACACUA -14.7 U U C UG U --------------------------------------------------------------------------------------------- miR-308 GUGU GGACACUAA -13.2 GA CAUAUUA --------------------------------------------------------------------------------------------- skl271_vir GU----GAAGGCUGAUUA-AAAUGUUUGGAUAAGCGCAAAUGUGAUAAAUAAUUUUGCUAAAACAU skl271_moj GUUUUUGAAGGCUGAUUUGAAAUGUUUGGAUAAGCGCAAAUGUGAUAAAUAAUUUUGCUAAAAAUU skl271_pse AAUUGCCGUCGC-GAUACACACUUUUUGGAUAAGCGCAAAUGUGAUAAAUAAUUUUGCUAAAAAUA skl271_ana CU--------------UAAAACACUUUGGAUAAGCGCAAAUGUGAUAAAUGAUUUUGCUAAAAAUA skl271_yak -------------------AAAACUUUGGAUAAGCGCAAAUGUGAUAAAUAAUUUUGCUAAAAAAA skl271_mel -------------------AAAACUUUGGAUAAGCGCAAAUGUGAUAAAUAAUUUUGCUAAAAAAA

987654321 miR-6 UCU UCG GU ACACUAU -20.3 UUUU UG G --------------------------------------------------------------------------------------------- mir-2b GAGG UUCG ACACUAU -17.0 C AGU ACCG --------------------------------------------------------------------------------------------- miR-11 UUGAG UCUG ACACUA -12.3 UCGUUC C --------------------------------------------------------------------------------------------- miR-308 UC UAUU ACACUA -11.4 GAGUG A GGA A --------------------------------------------------------------------------------------------- miR-13b GAGC GUUU CG ACACUAU -11.9 U A UAC --------------------------------------------------------------------------------------------- Sequences surrounding the target sites for the 6 available Drosophilid species (D. melanogaster, D. yakuba, D. ananassae, D. pseudoobscura, D. virilis, and D. mojavensis) were retrieved from VistaBrowser (http://pipeline.lbl.gov/cgi-bin/gateway2) and aligned, species are ordered by distance from D. melanogaster; nucleotides conserved in all species are shaded blue, nucleotides conserved in the majority are shaded gray. Target sites are named according to the position in the 3′UTR that is complementary to the 5′ end of the miRNA (position 1), in D. melanogaster coordinates. Pairings of miR-2 family miRNAs with the target sites are based on optimal foldings as computed with mfold (Zuker, 2003) and are listed in order of ∆G value. Watson-Crick base pairings are shaded red, G:U pairings yellow, nonpairing residues of the miRNA are offset. For miR-2 and miR-13, only the miRNA copy with the best alignment is shown.

Supplemental References Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.