SUPPLEMENTARY MATERIAL Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility Dewi Astuti (1)*, Mark R Morris (1,2)*, Wendy N Cooper (1), Raymond H.J. Staals (3), Naomi Wake (1), Graham A Fews (4), Harmeet Gill (1), Dean Gentle (1), Salwati Shuib (1), Christopher J Ricketts (1), Trevor Cole (4), Anthonie J. van Essen (5), Richard van Lingen (6), Giovanni Neri (7), John M Opitz (8), Patrick Rump (5), Irene Stolte-Dijkstra (5), Ferenc MÜller (1), Ger J.M. Pruijn (3), Farida Latif (1), Eamonn R Maher (1,4) 1. Centre for Rare Diseases and Personalised Medicine, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK 2. School of Applied Sciences, University of Wolverhampton, Wolverhampton, WV1 1LY, UK 3. Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands 4. West Midlands Region Genetics Service, Birmingham Women s Hospital, Edgbaston, Birmingham B15 2TG, UK 5. Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 6. Department of Pediatrics, Isala Clinics, Zwolle, The Netherlands, 7. Istituto di Genetica Medica, Università Cattolica del S. Cuore, Rome, Italy 8. Departments of Pediatrics, Human Genetics, Obstetrics, and Gynecology, University of Utah, Salt Lake City, UT, USA *Contributed equally Correspondence to: Professor Eamonn R Maher, Centre for rare Diseases and Personalised Medicine, Institute of Biomedical Research (West), University of Birmingham, The Medical School, Edgbaston, Birmingham B15 2TT, UK Email E.R.Maher@bham.ac.uk Telephone number 0121 627 2741
PLK1-RT-F 5 -CAAGTACGGCCTTGGGTAT-3 PLK1-RT-R 5 -CTTGGTGTGATCCTGGAAG-3 AuroraKB-RT-F 5 -CAGCCACGATCATGGAG-3 AuroraKB-RT-R 5 -GTCCACCTTGACGATGC-3 PLK4-RT-F 5-ATGCTAATGAGGGTCATCGT-3 PLK4-RT-R 5 -GCTCCACTAGTTAACTGTGTAGCC-3 TKK-RT-F 5 -AGGTTCAAGCAAGGTATTTCAG-3 TKK-RT-R 5 -TGTGAACAATGCCATGTTG-3 GAPDH-RT-F 5 -GACCCCTTCATGACCTCAACTACA-3 GAPDH-RT- R 5 -CTAAGCAGTTGGTGGTGCAGGA-3 DIS3L2-A-F 5 -GCAGTGGAGGTTTCTCTGGA-3 DIS3L2-A-R 5 -CCTCCTTCCAGTCCACAATG-3 DIS3L2-B-F 5 -ACCGGCTTCCTCAAACTCTT-3 DIS3L2-B-R 5 -GTACCTCCTCGCTGCTATGC-3 DIS3L2-C-F 5 -CGCTCCTGCACCAAACTTAG-3 DIS3L2-C-R 5 -GGCGAGGTGAAGTGTGTGTA-3 DIS3L2-D-F 5 -GCAGGAGCCCTCAATAAAAG-3 DIS3L2-D-R 5 -AGCTCAGCTGGTGCTTGAGT-3 DIS3L2-Not1-F 5 -GATGCGGCCGCGATGAGCCATCCTGACTACAGAATG-3 DIS3L2-BamH1-R 5 -GATGGATCCTCAGCTGGTGCTTGAGTCCTCGGGCTC-3 DIS3L2-Del6- BamH1-R 5 -GATGGATCCTTAGTATTCCTTCTGTTTCAGGCTCAA-3 Supplementary,Table,1:,Oligonucleo6de,and,primers,used, in,this,study.!!! Primers,for,RT>PCR,,,cDNA,amplifica6on,and,DIS3L2,constructs,genera6on!
DIS3L2-2F 5 -CCAGAGAATGTGATGCCAGA-3 DIS3L2-2R 5 -CTCCACTTCACACCCATTCC-3 DIS3L2-3F 5 -GGGAGTTTCAGTCTCTTGTTGG-3 DIS3L2-3R 5 -AGCCCTGCACCTGTTACATC-3 DIS3L2-4F 5 -CATAAATTGTGTTTCCTGCATTT-3 DIS3L2-4R 5 -TGATTTCAGTTTCGGAGCAG-3 DIS3L2-5F 5 -TCAGCATGCTTTATTTAATACGAA-3 DIS3L2-5R 5 -TGGAATGTGAAAGACCCTAAGAA-3 DIS3L2-6F 5 -TGGTCTGTGCTTCTGGAAAA-3 DIS3L2-6R 5 -CAGCAGAGAGGGAATTCCAATA-3 DIS3L2-7F 5 -TGAGTAGTTTGGGGTAACTGGTA-3 DIS3L2-7R 5 -CCTGACTGAATCACACGAGA-3 DIS3L2-8F 5 -AAAAACTGCTGGTTCCTGCT-3 DIS3L2-8R 5 -TCTGCAAGGAGAGGACTGGT-3 DIS3L2-9F 5 -AACGTACAGGCACTCTGAAACT-3 DIS3L2-9R 5 -TAGCCTGTGGGCCATAATTT-3 DIS3L2-10F 5 -TCACTTTCCCTAACAGAGCACA-3 DIS3L2-10R 5 -ATGCAGACTGCCTGCTACC-3 DIS3L2-11F 5 -GTAGTGCGGGTCACAGAGG-3 DIS3L2-11R 5 -TTCCTTTGGCTTCTCTTTCC-3 DIS3L2-12F 5 -ACTCTAGAGGCGCATGGAAA-3 DIS3L2-12R 5 -GCACATGGTGAGTGCTTCAT-3 DIS3L2-13F 5 -CATTCCGAGCACACAGTCAG-3 DIS3L2-13R 5 -GTGCCTGGAGCAAAGAGAAG-3 DIS3L2-14F 5 -ACGTATTGGTTGCAGCTGA-3 DIS3L2-14R 5 -AGGGGCAAAGAGATCTTACAA-3 DIS3L2-15F 5 -AGCATGGTGCCCTCTTACAG-3 DIS3L2-15R 5 -ACAGGAGAGACCTGGCTGAA-3 DIS3L2-16F 5 -GCCAGCAAAAACATGGAAGT-3 DIS3L2-16R 5 -ACAGGTCTGGGGCTAGTGTG-3 DIS3L2-17F 5 -CTCCCTGGCGTCACTCAG-3 DIS3L2-17R 5 -CTGTCACGGCAGAATGGTC-3 DIS3L2-18/19F 5 -GGGAGTGAGCCCTCATGATA-3 DIS3L2-18/19R 5 -CTGGCAGAAATGAGGCTTTG-3 DIS3L2-20F 5 -AGGGCCTCCCTGAAGCTC-3 DIS3L2-20R 5 -GGAGAGGGGTCATCAGGACT-3 DIS3L2-21F 5 -CATTACAGACAGGCGAGCAG-3 DIS3L2-21R 5 -GCAGTAGGGCCCAGAAAAA-3 Primers,for,amplifica6on,of,DIS3L2,exons,for,muta6on,analysis! Zfdis3l2-pCS2-F 5'-GATTCGAATTCAAGGATGGCATTTTCCCCTAA-3 Zfdis3l2-pCS2-R 5'-TAGAGGCTCGAGAGGTTACATGGGTGCTAAGG-3' Zfdis3l2-ATG MO 5 -ATGCCATAATCTCGTCTACAGTTAC-3 Zfdis3l2-UTR MO 5'-CTTGTCTATTTCACTGAACACTTCT-3' Zfdis3l2-mm MO 5'-CTTcTCTAaTTCAgTGAAgACTTgT-3' Primers,for,dis3l2'expression,in,zebrafish,and,zdis3l2,morpholinos!
Primers,for,MLPA,analysis,! Ex2-LPO Ex2-RPO Ex3-LPO Ex3-RPO Ex4-LPO Ex4-RPO Ex5-LPO Ex5-RPO Ex6-LPO Ex6-RPO Ex7-LPO Ex7-RPO Ex9-LPO Ex9-RPO Ex10-LPO Ex10-RPO Ex11-LPO Ex11-RPO Ex12-LPO Ex12-RPO Ex13-LPO Ex13-RPO Ex14-LPO Ex14-RPO Ex15-LPO GGGTTCCCTAAGGGTTGGAGAGTCTCTAAGCAGTGGAGGTTT CTCGGATCTGGAGAG P -AAGAGTGACCTTGGAGCCAATAATGAGCCATCCGTCAGC TCTAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGACGGACATTGGTGCTTCGCCAGGT GACAAAA P -AGTCAAAGAACAGGTCCACACGAGGGAATTCTAGATTGG ATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGATGCAAAGAGCAGCTGGCCCAGG CAGA P -CTACCCACCAGTTCGTGTAACTGGACGTCTAGATTGGATC TTGCTGGCAC GGGTTCCCTAAGGGTTGGAGTCGTGGGTTGTTGCTCGTAATA GAGCCTTAAATGG P GGATCTGGTGGTCGTGAAACTGCTTCCATCTTTCTAGATT GGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGACTCTTCTCAGTTTGATGGCAGCG ACTCAGAAGATGGA P CATGGCATCACACAAAATGTGCTGGTTGATGGGTGGTCT AGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGAGGGGCCGTGCTTCCGAGAGGAT GGTGATGCACCGGTTACAAAAGAT P GAGACCACCTGCATTTCACAAGACACAAGAGCGGGCGAT TCTCCTCTAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGATGGCTAGAATGTCTTCCTCAAGG CCTGCCA P TGGACAATTCCACCAGAGGAGTTCAGCCCTCTAGATTGGA TCTTGCTGGCAC GGGTTCCCTAAGGGTTGGAGACCTCGATGATGCCCTCTCCTG CAAG P CCACTCGCTGACGGTAGGATGGAAATTTCTAGATTGGATC TTGCTGGCAC GGGTTCCCTAAGGGTTGGATGGACCATTGCTGACGTGAGTTA CTTTGTTCCGGAGG P GATCTGATCTGGATAAAGTGGCTGCCGAGAGGTAGCTCT AGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGAGACCAAACATGTCCGACAAGCT GACCTTCTCTGTGATCT P GGACACTGACTCCAGAGGGCAAGGTAACAACTGCACATG TCTAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGAGCTAGTAAAAAACGGGAACCAG AGCATAGCAGCGAGGAGGTACAC P CAGGCCGTCTTGAATCTCCACGGAATTTAACCTACTGGCA TAACTTCTAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGACGAGGACTAGCCCTAGGTAGTTC ACTCTGGACCACGAGACCGGATTG P CCTCAAGGATGTCATATCTATGAGTACCGCGCTCTACCAC CTGCCTAACATCTAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGATGATTGGAGGAGTTCATGCTCTT GGCCA
Ex15-RPO Ex16-LPO Ex16-RPO Ex18-LPO Ex18-RPO Ex19-LPO Ex19-RPO Ex20-LPO Ex20-RPO Ex21-LPO Ex21-RPO Primers,for,MLPA,analysis,con6nued,! P ACATGGCAGTGGCCCACAAGATAACATCTAGATTGGATC TTGCTGGCAC GGGTTCCCTAAGGGTTGGATGGAGCTGTGAGTCCAGGCCAG CCAGCAAAA P ACATGGAAGTGTGGGCCACAGGGTTGCCTCTAGATTGGAT CTTGCTGGCAC GGGTTCCCTAAGGGTTGGATGAACCTATCGTGCAGGAGCTCA GTACCAGTCTCTTC P TTTGCTGTTCTGGTCAAGGTGAGCCCTCACTAAGTTTCTA GATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGATCTCTGTTAAAAATGGAGTCAGA AGCCATGGTGATGGGCA P TCCTGAAGCAAGCCTTCGACGTGCGTTGTAGTATAGCATC TAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGACCAGAACTTACAGTTAAAGTATT TCCCACCACTTCCAGAAGGTGG P GCAAGAAGCCGGAACTCACGCTGGTAGTTTCTAAGTCTT CACGTTTCTAGATTGGATCTTGCTGGCAC GGGTTCCCTAAGGGTTGGAGATTAACTTACAAACTTTAACAA GTTGCCCTGAGAAGGAGGAGGAGGAGTC P TGACGGTGAGCCCGAGGACTCCCTACAACTAGTCTAGAA CAACTAATCTAGATTGGATCTTGCTGGCAC
Supplementary,Figure,1.!Upper%panel:!Clinical!photographs!of!Perlman!syndrome! pa7ent! P4.1! showing! craniofacial! dysmorphisms!(everted! upper! lip,! broad! nasal! bridge).!lower%panel:!family!pedigrees!for!p1bp5.!consanguinity!is!indicated!for!p1! and!possible!consanguinity!for!p2!and!p3.!
Supplementary, Figure, 2:, Known, genes, and, puta6ve, transcripts, located, within, the, 2.1, Mb, minimal, region, of, homozygosity., (a)! Genes! and! transcripts! were! iden7fied!by!inspec7on!of!the!ucsc,!ncbi!and!ensembl!databases,!arrow!heads! indicate!the!direc7ons!of!transcrip7on!(due!to!constraints!of!the!scale!not!all!exons! of! all! genes! are! depicted).!(b)! Diagram! of! DIS3L2! protein! showing! the! loca7on! of! the!predicted!rnb!domain,!and!the!loca7ons!of!the!muta7ons!found!in!individuals! with!perlman!syndrome.!
Homo_sapiens -------------------------------------------------- Mus_musculus -------------------------------------------------- Monodelphis_domestica ----MDHLGFVRSGVREGTSKLAVPQKNAYARLVQQHRSGRFRSYLNHVA 46 Xenopus_tropicalis -------------------------------------------------- Danio_rerio MAFSPKAKRTPKNKDGKGAQNLPKPQKDAYSRLLNQHQNGKFRSYLQEFV 50 Arabidopsis_thaliana ---------MKSASSEQSVERIENGHKKKRNRPQKQNRRSKQSS------ 35 Drosophila_melanogaster -MSETSSVNVDADRDCRRIEGLRQRSQRLAARLQQMQREMESNASSKDLS 49 Homo_sapiens -------------------------------------------------- Mus_musculus -------------------------------------------------- Monodelphis_domestica QKMQLEEMGLADAGSDADLPLKVGMTREAVTRNGLNVPGSSPKKAIPPDS 96 Xenopus_tropicalis -------------------------------------------------- Danio_rerio EQQSEENATTSVSRSDANR-RKKGHSESSYFGDSVSSPQSRPMLADTFNS 99 Arabidopsis_thaliana --VPIEDAHVEESLDGRDSSRSKAKDSTSSSKQ----------------- 66 Drosophila_melanogaster ANNAVIPLQPLMRWAEPRPHPNQSQPSARSNSRQAIPYGVPVSTPAPVAM 99 Homo_sapiens -------------------------------------------------- Mus_musculus -------------------------------------------------- Monodelphis_domestica TECLGVEMLPDDSLEASRKAPEMCPLGVGEMKEAYCSHPGESIFEVESLS 146 Xenopus_tropicalis ------------------------------------MHNSEFLSPVQSGT 14 Danio_rerio ------------------------------------FHADKTFHQVTCAE 113 Arabidopsis_thaliana ------------------------------------QRPNTDELEAMRAS 80 Drosophila_melanogaster QLP-----------------------------YHVAFASYQATMQIPCLQ 120 Homo_sapiens -------------------------------------------------- Mus_musculus -------------------------------------------------- Monodelphis_domestica RGLSGCKFAQSPRSGHVLQKAGASVDKDVSETGQSRLQKGKYATNVEKAR 196 Xenopus_tropicalis QRGTNRSILN---------------------------------------- 24 Danio_rerio DPGTNPGMLEKLHKGSAPKKASAKLKKSEKDIKASTSLVGSEEGDAQQL- 162 Arabidopsis_thaliana NVAFNSMPPMRAESG----------------------------------- 95 Drosophila_melanogaster PLSHLHVVSSPSHRLIAKKKSKRALRNEVEALQDMVKHLSHLSPDVN--- 167 Homo_sapiens -------------------------------------------------- Mus_musculus -------------------------------------------------- Monodelphis_domestica DISVAEERQCHLDPHSLTSERPGPLPPEQLPGSRPRKSSELSSTPSNLGK 246 Xenopus_tropicalis -------------------------------------------------- Danio_rerio ---------------------------------SGSCGEEKTRKKSKKKS 179 Arabidopsis_thaliana -------------------------------------------------- Drosophila_melanogaster -----------------------------------------------AYA 170 Homo_sapiens ---------MSHPDYRMNLRPLGTPRGVSAVAGPHDIGASPGDKKSKNRS 41 Mus_musculus ---------MNHPDYKLNLRSPGTPRGVSSVVGPSAVGASPGDKKSKNKS 41 Monodelphis_domestica KRQLLEAESMSHLDCRTHPKQLGTPKGVSHALGCSGTGSMPGDRKMPSKH 296 Xenopus_tropicalis ----------------------------------------------NKKS 28 Danio_rerio SKGHLEKKDKGACGLEVDAAEKSANKNVKASQALKGSRQI-DKKDINAKD 228 Arabidopsis_thaliana ------YPRRSASPLLSSPEVSKQLLSKSCPDPRACEQSPGMNGELFQQI 139 Drosophila_melanogaster CQLIRKNEILSQLEWSQTRSEAHPQQVGYQGRHRHDSEVSATNNGSHVQQ 220 : Homo_sapiens TRGKKKSIFETYMSKEDVSEGLKRGTLIQGVLRINPKKFHEAFIPSPDGD 91 Mus_musculus MRGKKKSIFETYMSKEDVSEGLKRGTLIQGVLRINPKKFHEAFIPSPDGD 91 Monodelphis_domestica SQGKKRSVFESYMSKESVSAGLKRGTLIQGVLRINPKKYHEAFIPSPDGT 346 Xenopus_tropicalis GKGKKKSVFEAYMTKEEVSAGLKRGELIQGPLRINPKKFHEAYLPSPDGV 78 Danio_rerio GRASGKQVFEQYMSVEDVSAGLKRGELIQGVLRINPKKYKEAYIPSPDGS 278 Arabidopsis_thaliana EGSSQRKIFSSHWSLDAVTEALEKGEAFKALFRVNAHNRNEAYCKIDGVP 189 Drosophila_melanogaster QPKLGRRQRNSKKNDGSGSNGTQSQKTELEAMRSYINKIVQRHVGMDHQL 270 :.. :. : :* :: :. Homo_sapiens -RDIFIDGVVARNRALNGDLVVVKLLPEEHWKVVKPESNDKETEAAYESD 140 Mus_musculus -RDIFIDGVVARNRALNGDLVVVKLLPEDQWKAVKPESNDKEIEATYEAD 140 Monodelphis_domestica -RDIFINGVVARNRALNGDVVVVELLPKDRWKVIKTDDSDKGGDTVPESD 395 Xenopus_tropicalis -RDLFIDGVVPRNRALNGDVVVVKLLPQEQWKVLKNDVCEDDDTPGHSTG 127 Danio_rerio -ADIFLDGISARNRALNGDVVVVKVLPLEQS------------------- 308 Arabidopsis_thaliana -TDILINGNVCQSRAVEGDTVVIKLDPLSLWPKMKGFVTESAAKPEGTNS 238 Drosophila_melanogaster SEDLIFKGNFSDLEAHAQRLVAAGYGRIVEEEIRVNRKNNRQAFIIMSTD 320 *:::.*.* *. Homo_sapiens IPEELCGHHLPQQSLKSYNDSPDVIVEAQFDGSDSEDGHGITQNVLVDGV 190 Mus_musculus IPEEGCGHHPLQQSRKGWS-GPDVIIEAQFDDSDSEDRHGNTS-GLVDGV 188 Monodelphis_domestica VTEELLRTPPQQLGRRCTADSPDVILEAQLNDSETEDGQ-------EHGP 438 Xenopus_tropicalis NKQHALSPHLM----KSSAKNPDLIIEAKVDSSA------------EDGH 161 Danio_rerio ----------------QATKTPDVIVEAHYNGDD------------EMVS 330
Arabidopsis_thaliana PPEKDDKKARQKNGIDVVEGFEDGFSKNKSSVIGKGAKNGVTPSSPPSLD 288 Drosophila_melanogaster REALERDGIVLLPVARRYAFDGDKVRAFVLNPGAQGSSKTAEPSSGEISG 370 *.. Homo_sapiens KKLSVCVSEKG----REDGDAPVTKDETTCISQDTRALSEKSLQRSAKVV 236 Mus_musculus KKLSISTPDRG----KEDSSTPVMKDENTPIPQDTRGLSEKSLQKSAKVV 234 Monodelphis_domestica SVLAEVTQEKG----ENDPSIPMLSSGNSPKSQDTRLCPDNLLQRSAKVV 484 Xenopus_tropicalis ESALIGCLQKE----IKDQDKLGAIEEKTSKQGDPKTFSDDCFQKTAKVV 207 Danio_rerio KMEKVSLNDKA----LLTTQSV----EHATTSNTPASSSERTTQKNGKVV 372 Arabidopsis_thaliana SCLGSFCEQKGNCSAVDKLCGILSSFPHKRPTGQVVAVVEKSLVRDSIVG 338 Drosophila_melanogaster GKPSLSLADGE------ELSDDTESQGSESDTDNVVVISSDNCPK-AFVI 413 :. :. * Homo_sapiens YILEKKHSRAATGFLKLLADKNSELFRKYALFSPSDHRVPRIYVPLKDCP 286 Mus_musculus YILEKKHSRAATGILKLLADKNSDLFKKYALFSPSDHRVPRIYVPLKDCP 284 Monodelphis_domestica YILEKKHSRAATGFIKPLADKNSELFRKCALFSPSDHRVPRVYVPLADCP 534 Xenopus_tropicalis YILEKKHSRAATGFIKPLSDKSSDLARKRALFSPVDHRLPRIYVPLGDCP 257 Danio_rerio FIVEKKHSRAVSGFLKFLPNK------RFAMFSPVDHRVPRVNVPLADCP 416 Arabidopsis_thaliana LLDVKGWIHYKESDPKRCKSPLSLSDDEYVQLMPADPRFPKLIVPFHVLP 388 Drosophila_melanogaster AITKRTELRQIVGTISFTNPTKLCDDQLFYKFRPYDLRVPMVYVPKDACA 463 : : :.. : * * *.* : **. Homo_sapiens QDFVARPK----DYANTLFICRIVDWKEDCNFALGQLAKSLGQAGEIEPE 332 Mus_musculus QDFMTRPK----DFANTLFICRIIDWKEDCNFALGQLAKSLGQAGEIEPE 330 Monodelphis_domestica ADLVSRPN----DYAQALFVCRIVAWEEHSHFAVGHLAKSLGQAGEIEPE 580 Xenopus_tropicalis HDFAIHPE----TYANTLFICSITAWRDDSNFAEGKLMKSLGQAGEIEPE 303 Danio_rerio ADFTSRPG----DYENTLFICRITQWPADSNFAEGRLAKTLGQAGEIEPE 462 Arabidopsis_thaliana GSIRARLENLDPNLEAELVAAQIVDWGEGSPFPVAQITHLFGRGSELEPQ 438 Drosophila_melanogaster AHIGNKQQ---IDVSGLLYLAHILETDCNG-HCIAELIQPVGRVGNLDDE 509 : : *. *...: :.*:.::: : Homo_sapiens TEGILTEYGVDFSDFSSEVLECLPQGLPWTIPPEEFSKRRDLRKDCIFTI 382 Mus_musculus TEGILTEYGVDFSDFSSEVLECLPQSLPWTIPPDEVGKRRDLRKDCIFTI 380 Monodelphis_domestica TEGILMEYGVDFSDFSPEVLECLPQALPWTIPPAEFSKRRDLRKDCVFTI 630 Xenopus_tropicalis TEGILVEYGVDFSDFPDKVLQCLPQDLPWTIPQEEFQKRKDLRNECIFTI 353 Danio_rerio TEGILTEYGVDFSEFTEEVLNCLPQDLPWSIPAHELSTRRDLRKECIFTI 512 Arabidopsis_thaliana INAILYQNSVCDSDFSPGSLTSLPR-VPWEVPEEEVQRRKDLRDLCVLTI 487 Drosophila_melanogaster LKAILFHNGLRDIKPFEQRFIDIYSQPPPPISQEDLRQRKDLRKMCIFTI 559 :.**..:. : : * :. :. *:***. *::** Homo_sapiens DPSTARDLDDALSCKPLADGNFKVGVHIADVSYFVPEGSDLDKVAAERAT 432 Mus_musculus DPSTARDLDDALACRRLTDGTFEVGVHIADVSYFVPEGSSLDKVAAERAT 430 Monodelphis_domestica DPATARDLDDALSCRPLPDGNFEVGVHIADVSYFVPEGSALDKVASERAT 680 Xenopus_tropicalis DPATARDLDDALSCKPLPDGNFEVGVHIADVSYFVAEGSALDIMASERAT 403 Danio_rerio DPATARDLDDALSCKQLSDGNFEVGVHIADVSYFVKEGNALDELAGQRAT 562 Arabidopsis_thaliana DPSTATDLDDALSVQSLPGGFFRVGVHIADVSYFVLPETALDTEARFRST 537 Drosophila_melanogaster DPMTARDLDDAVSIEKLGDNEYEIGVHISDVSHFLIEDNELDNIVKERST 609 ** ** *****::. *.. :.:****:***:*:. **. *:* Homo_sapiens SVYLVQKVVPMLPRLLCEELCSLNPMSDKLTFSVIWTLTPEGKILD--EW 480 Mus_musculus SVYLVQKVVPMLPRLLCEELCSLNPMTDKLTFSVIWKLTPEGKILE--EW 478 Monodelphis_domestica SVYLVQKVVPMLPRLLCEELCSLNPMTDKLTFSVVWKLSPEGKILD--EW 728 Xenopus_tropicalis SVYLVQKVIPMLPRLLCEELCSLNPMTDRLTFSVIWKITPQGEILD--EW 451 Danio_rerio SVYLVQKVIPMLPRLLCEELCSLNPLTDRLTFSVIWKLSPEGKILS--EW 610 Arabidopsis_thaliana SVYLMQRKISMLPPLLSENVGSLSPGADRLAFSILWDLNREGDVID--RW 585 Drosophila_melanogaster SIYLANEVIHMLPQSLCMR-CSLLPGQDKFAFSVFWRMNGKGVMLQKKPE 658 *:** :. : *** *.. ** * *:::**:.* :. :* ::. Homo_sapiens FGRTIIRSCTKLSYEHAQSMIESPTEKIPAKELPPISPEHSSEEVHQAVL 530 Mus_musculus FGRTIIRSCTKLSYDHAQSMIENPTEKIPEEELPPISPEHSVEEVHQAVL 528 Monodelphis_domestica FGRTIICSCTKLSYDHAQSMIEHPDKKFHEDELPPVSTDHPIEQIHQAIL 778 Xenopus_tropicalis FGRSVICSCVKLSYDHAQNMINHPDKKIEQHELPPVSPQHTINEIHQAVL 501 Danio_rerio FGRTVICSCIKLSYDHAQSMIDAPDKHFSPEELPACSPNHSIIEIQEAVL 660 Arabidopsis_thaliana IGRTIIRSCCKLSYDHAQDIIDGKSD-VAENGWPALHGSFKWCDVTRSVK 634 Drosophila_melanogaster FCRTVINSCSQFAYEHAQKIIDNPNERFTENDFPTILNGFNPDDIRNRVL 708 : *::* ** :::*:***.:*:... *.. ::. : Homo_sapiens NLHGIAKQLRQQRFVDGALRLDQLKLAFTLDHETGLPQGCHIYEYRESNK 580 Mus_musculus NLHSIAKQLRRQRFVDGALRLDQLKLAFTLDHETGLPQGCHIYEYRDSNK 578 Monodelphis_domestica NLHGIATHLRRQRFVDGALRLDQLKLAFTLDQDSGMPQGCHIYEYKDSNK 828 Xenopus_tropicalis NLHLIAQNLRKQRFDDGALRLDQLKLTFTLDKESGLPQGCYIYQYRDSNK 551 Danio_rerio NLHNIAKQLRAQRFEGGALRLDQLKLSFTLDKESSMPQGCYVYQYRDSNK 710 Arabidopsis_thaliana QLSEISTTLRQKRFRNGALQLENSKPVFLFD-EHGVPYDFVTCSRKGSNF 683 Drosophila_melanogaster WLHDIASSIRKTRLDNGALTINNAKLRFLLDPITGEPLSFEVEKQREANR 758 * *: :* *:.*** ::: * * :*. *.. : :* Homo_sapiens LVEEFMLLANMAVAHKIHRAFPEQALLRRHPPPQTRMLSDLVEFCDQMGL 630
Mus_musculus LVEEFMLLANMAVAHKIFRTFPEQALLRRHPPPQTKMLSDLVEFCDQMGL 628 Monodelphis_domestica LVEEFMLLANMAVAHQIYRAFPERALLRRHPPPQSKLLSDLEELCDQLGL 878 Xenopus_tropicalis LVEEFMLLANMAVAHHIYRRFPEEALLRRHPPPQTKMLNDLIEFCDQMGL 601 Danio_rerio LVEEFMLLANMAVGHQIYRSYPELALLRRHPPPQTKMVDDMQEFFDQMGL 760 Arabidopsis_thaliana LVEEFMLLANMTAAEVISQAYRASSLLRRHPEPNTRKLKEFEGFCSKHGM 733 Drosophila_melanogaster LIEEFMLLANQAVARFIHDSFPDIAVLRNHPPPLIKSLKALREKLLALGF 808 *:******** :... * : ::**.** * : :. : *: Homo_sapiens PVDFSSAGALNKSLTQTFGDDKYSLARKEVLTNMCSRPMQMALYFCSGLL 680 Mus_musculus PMDVSSAGALNKSLTKTFGDDKYSLARKEVLTNMYSRPMQMALYFCSGML 678 Monodelphis_domestica AIDFSSAGALHKSLTEMFGDDMYSLAWKEVLTNMCSRPMQ---------- 918 Xenopus_tropicalis QLDFTSSGTLHKSLNDQFETDEYSAARKEVLTNMCSRPMQMAVYFCTGAL 651 Danio_rerio DIDFSSSGALHRSLNENLGDDEYTAARKEVITHMCSRPMQMAVYFCAGAL 810 Arabidopsis_thaliana DLDISSSGQLQDSLEKITGNLKDDSVFVDILNNYAIKPMQLASYFCTGNL 783 Drosophila_melanogaster ELDYSSSKALQESMVRLCNEAPNPVAMNACLSQLLMKPMARATYFCSEGK 858 :* :*: *: *:. :.: :** Homo_sapiens QD-PAQFRHYALNVPLYTHFTSPIRRFADVLVHRLLAAALGYRERLDM-- 727 Mus_musculus QD-QEQFRHYALNVPLYTHFTSPIRRFADVIVHRLLAAALGYSEQPDV-- 725 Monodelphis_domestica -------------------------------------------------- Xenopus_tropicalis KD-ETLFHHYALNVPLYTHFTSPIRRFADVIVHRLLAASLGCGPPLKM-- 698 Danio_rerio KD-EKLFRHYALNVPLYTHFTSPIRRYADVIVHRLLGASLNCGPRLSV-- 857 Arabidopsis_thaliana KDSVAEWGHYALAVPLYTHFTSPLRRYPDIVVHRALAAALEAEELYSKQK 833 Drosophila_melanogaster SE-PADLWHYALSIPIYTHFTSPIRRYPDILVHRLLAAALKYCTPPKR-- 905 Homo_sapiens ----------------------------------------APDTLQKQAD 737 Mus_musculus ----------------------------------------EPDTLQKQAD 735 Monodelphis_domestica -------------------------------------------------- Xenopus_tropicalis ----------------------------------------PKEVIQKQAD 708 Danio_rerio ----------------------------------------SGEEVHQQAS 867 Arabidopsis_thaliana QTAIDEGRSCFTGIHFNKDAAESIEGKEALSVAALKHGVPSTEILSDVAA 883 Drosophila_melanogaster ----------------------------------------TPDDLHTLTK 915 Homo_sapiens HCNDRRMASKRVQELSTSLFFAVLVKESGPLESEAMVMGILKQAFDVLVL 787 Mus_musculus HCNDRRMASKRVQELSIGLFFAVLVKESGPLESEAMVMGVLNQAFDVLVL 785 Monodelphis_domestica -------------------------------------------------- Xenopus_tropicalis HCNDRKTASKRVQELSAELFFSVFVKECGPLESEAMVMGVLNEAFDVIVL 758 Danio_rerio HCNDRKTASKRVQELSSELFFAVFVKECGPLDSEAMVMGVLDQSFDVLVL 917 Arabidopsis_thaliana YCNERKLAARKVRDACDKLYTWFVLKQKEIFPCEARVMNLGSRFMTVYIS 933 Drosophila_melanogaster LANERKYNAKKAGEDSGNLYFKRYVHNKQGIYMRAVVIEIFQHMMNVVTL 965 Homo_sapiens RYGVQKRIYCNALA-LRSHHFQKVGKKPELTLVWEPEDM-EQEPAQQVIT 835 Mus_musculus RFGVQKRIYCNALA-LRSYSFQKVGKKPELTLVWEPDDL-EEEPTQQVIT 833 Monodelphis_domestica -----------ALP-LQNHHFKKVGKKPELTLIWEPESP-QQKAVPQVIT 955 Xenopus_tropicalis RFGVQKRIYCNSLP-LVNSHFQQVGKRPELTLLWEPEKK-GEEPVRQVIS 806 Danio_rerio RYGVQKRIYCNSLEGLKEVEFRKVGKKPEMTLVWETDDP-SKDSIKQEIS 966 Arabidopsis_thaliana KLGIERRIYYDQIEGLCADWLEATSTLIVDKLYSKRGGRGFFKPMKEAVY 983 Drosophila_melanogaster ESG-----HVISINYKMQKVLVDTHGVPNYILIAERNLK-QSPRKLQLLS 1009 : :. * : : : Homo_sapiens IFSLVEVVLQ------------AESTALKYSAILKRPGTQGHLGPEKEEE 873 Mus_musculus IFSLVDVVLQ------------AEATALKYSAILKRPG---------LEK 862 Monodelphis_domestica IFSLVEVVLE------------AEDDTLKYVATLKRPE---------GTQ 984 Xenopus_tropicalis IFTLVEVVLK------------SDNVPLKYTAVLKSPE------------ 832 Danio_rerio IFTLLNVQLK------------ADGAPMKYSATLKRPL------------ 992 Arabidopsis_thaliana LVSPCEVCVAKCSALSVHDTESPEAVSIDEVAPAVFPLTIQLFSTIPVVL 1033 Drosophila_melanogaster VVPICLIIWD-----------------QKLTGFLKMEEQI---------- 1032 :.. :.. Homo_sapiens ESDGEPEDSSTS---------- 885 Mus_musculus ASDEEPED-------------- 870 Monodelphis_domestica APEDGPRDRDG----------- 995 Xenopus_tropicalis AQ-------------------- 834 Danio_rerio APM------------------- 995 Arabidopsis_thaliana HAVGGDDGPLDIGARLYMSSYY 1055 Drosophila_melanogaster ----------------------
Supplementary,Figure,4, A HeLa:,exogenous/endogenous,DIS3L2,expression, Ab:!aBDIS3l2! Ab:!aBFLAG! Merge! B COS7:,exogenous,DIS3L2,expression, Ab:!aBDIS3l2! Ab:!αBFLAG! Merge! HeLa:,Endogenous, COS7:,Endogenous,, COS7:,Endogenous,,, Ab:!αBDis3l2! Ab:!αBDis3l2! Ab:!αBDis3l2! C D E CounterBstain:! αbtubulin!!
F wtdis3l2 G Ab: α-flag mtdis3l2 del9 DAPI Merge H Ab: α-flag mtdis3l2 del6 DAPI Merge Ab: α-flag DAPI Merge Supplementary,Figure,4.!Cellular,localisa6on,of,DIS3L2.,Exogenous!expression!of! DIS3L2!containing!an!NBterminal!FLAG!tag!in!HeLa!(A)!and!COS7!cells!(B,!F)!indicate! that! DIS3L2! is! predominantly! expressed! in! the! cytoplasm.! Immunofluorescent! staining! of! endogenous! DIS3L2! in! HeLa! (C)! and! COS7! cells! (D,! E)! verified! cyctoplasmic! localiza7on! of! DIS3L2.! An! exon! 9Bdele7on! mutant! localised! in! the! cytoplasm!with!a!similar!pa[ern!as!wtdis3l2!(g).!an!exon!6bdele7on!mutant!had!a! diffuse!pa[ern!of!expression!and!was!found!to!be!present!in!both!the!nucleus!and! the!cytoplasm!(h).!nuclear!dna!was!stained!with!dapi!(blue).!
A EV 120kDa B 120kDa wtdis3l2 Clones 1 3 4 120kDa C Del9-DIS3L2 Clones 1 3 4 5 D Del6-DIS3L2 Clones 2 3 5 28kDa Supplementary, Figure, 5.! Valida6on, of, polyclonal, Rabbit, an6>dis3l2, an6body, specificity.! Western! blots! of! HEK293s! transfected! with! an! empty! pflagbcmv4! vector! or! DIS3L2! constructs! probed! with! polyclonal! rabbit! an7! DIS3L2.! wtdis3l2! produced!a!single!band!at!110kda!(a,!b),!mt(δ9)dis3l2!transfected!cells!produced! a! band! at! 90kDa! (C),! mt(δ6)dis3l2! cells! produced! a! band! at! 30kDa(D).! 25μg! of! total!protein!was!loaded,!equal!loading!was!verified!by!india!ink!staining.!
RNA ct Ct Oligo hrrp40 KD DIS3L2 KD 7S rrna 5.8S rrna Supplementary,Figure,6.,DIS3L2,ac6vity,is,not,involved,in,5.8S,rRNA,matura6on., Hela! cells! were! transfected! with! RNAi! oligos! against! DIS3L2,! the! core! exosome! component!hrrp40!or!a!control!oligo.!total!rna!was!extracted!and!northern!blots! were!probed!with!a!cdna!probe!that!was!specific!to!7s!and!5.8s!rrna.!following!kd! of! hrrp40! the! immature! 7S! rrna! is! present.! However,! following! DIS3L2! KD! only! mature! 5.8S! rrna! is! present.! Knock! down! of! DIS3l2! was! validated! by! RTBPCR! and! Western!blohng,!Knock!down!of!hRRP40!was!validated!by!RTBPCR.!
EV! Wt!DIS3L2! 600 500 No.!of!cells! 400 300 200 100 0 1 2 3 4 5 6 7 No.!of!nuclei! Supplementary, Figure, 7.! Clones! of! the! RCC! cell! line! SKRC39! stably! expressing! wtdis3l2! were! found! to! contain! many! cells! with! either! mul7ple! dis7nct! nuclei! or! large!polyblobed!nuclei!(yellow!arrows).!for!analysis,!cells!were!grown!on!coverslips,! fixed!with!methanol!and!nuclear!content!labeled!with!ethidium!bromide.!
Supplementary,Figure,8, A, Number of interphase cells 200 150 100 50 3-4 centrosomes >5 centrosomes 0 ct Oligo DIS3L2 KD Supplementary, Figure, 8,(A)., DIS3L2, Knock, down, results, in, mul6ple, centrosome, forma6on., DIS3L2! was! knocked! down! in! HeLa! cells! using! Silencer! Select! sirnas! against!dis3l2!(s43418)!and!compared!to!cells!transfected!with!silencer!nega7ve! Control!#1!siRNA!(Ambion,!Aus7n,!TX,!USA).!4000!interphase!cells!were!scored!for! centromere!content!in!control!and!knockdown!cells.!23!control!cells!had!3!or!more! centrosomes!whereas!334!dis3l2!kd!cells!had!3!or!more!centrosomes,!156!of!these! cells! had! more! than! 5! centrosomes.! (B,, C), Pericentrin! and! (D), CDK5RAP2!! cob! localised!correctly!with!gammabtubulin!in!cells!that!had!mul7ple!centrosomes.!
Ct!RNAi!Oligo! DIS3L2!KD! GamaBTubulin! Pericentrin! Merge! Supplementary, Figure, 8, (B,C)., Pericentrin, coblocalised! correctly! with! gammabtubulin!in!cells!that!had!mul7ple!centrosomes.!!!
DIS3L!KD:!Pericentrin!localisa7on!! Supplementary,Figure, 8B, (Enlarged,for,clarity), GamaBTubulin! Pericentrin! Merge!
DIS3L!KD:!Pericentrin!localisa7on!! Supplementary,Figure,8, (C), GamaBTubulin! Pericentrin! Merge!
D, Ct!RNAi!Oligo! DIS3L2!KD! GamaBTubulin! CDK5RAP2!! Merge! Supplementary,Figure,8,(D),CDK5RAP2!coB!localised!correctly!with!gammaB tubulin!in!cells!that!had!mul7ple!centrosomes.!
Ct K.D. DIS3L2 K.D. E.V. wtdis3l2 A DIS3L2 B Cyclin D1 C p27 D p21 E Phospho-pRB prb Supplementary, Figure, 9., Dysregula6on,of,cell,cycle,control,proteins,following, DIS3L2,knock,down,or,over,expression.!Western!blot!analysis!was!preformed!72h! postbrnai!knock!down!of!dis3l2!by!silencer!select!sirnas!against!dis3l2!(s43418)! (DIS3L2!K.D.)!in!HeLa!cells!and!cells!transfected!with!a!control!RNAi!oligo!(ct!K.D.)!or! in!hek293!cells!stably!expressing!wtdis3l2!or!an!empty!vector!(a).!cyclin!d1!levels! were! elevated! following! KD! and! reduced! in! overbexpressing! cells! (B).! P27Kip1! expression! was! increased! in! cells! overbexpressing! DIS3L2! and! reduced! following! DIS3L2!KD!(C).!p21CIP1!was!upBregulated!in!cells!overBexpressing!DIS3L2!and!those! where!it!was!knocked!down!(d).!the!phosphoryla7on!state!of!prb!remained!in!its! inac7ve,! hyperphosphorylated! state! in! DIS3L2! KD! cells.! Whereas,! prb! was! found! predominantly! in! its! ac7ve,! cell! cycle! inhibi7ng,! hypophosphorylated! state! in! cells! overbexpressing! DIS3L2! (E).! 25μg! of! total! protein! was! loaded,! equal! loading! was! verified!by!india!ink!staining.!
Supplementary figure 10
Supplementary,Figure,11.,Dis3l2,is,required,for,normal,development,in,Danio'rerio! We!inves7gated!if!dis3l2!was!required!for!normal!zebrafish!development!by!knocking!down%dis3l2! with! morpholino! an7sense! oligonucleo7des!(mo).! Comparable! phenotypes! were! observed! with! two!independent!transla7on!blocking!mos:!morphant!larvae!injected!with!atg!mo!(a)!and!utr! MO!(B)!showed!mild!cardiac!oedema!(32%,!n=56!and!!!55%,!n=56!respec7vely)!as!compared!to! mismatch!mo!injected!controls!(c,!9.0%,!n=55)!while!no!obvious!signs!of!blood!circula7on!defects! were! detected.! Notable! morpholigical! defects! were! seen! in! the! development! of! the! pronephric! duct!opening!and!hindgut!around!and!including!the!cloaca!(d,e!as!compared!to!g).!the!region!was! either! disorganised! with! no! discernible! hindgut! and! pronephric! duct! opening,! or! forma7on! of! a! vesicle! instead! of! the! pronephric! duct! opening! was! seen.! As! addi7onal! targe7ng! specificity! control,!a!batch!of!atg!mo!injected!embryos!were!split!and!the!por7on!which!was!secondarily! injected! with! dis3l2! mrna! showed! recovery! of! the! cloaca! phenotype! (F,! 46,9%,! n=64),! as! compared! to! the! por7on,! which! was! secondarily! injected! with! control! cfp% mrna! (88%,! n=32,! P=0.0001)!while!the!mismatch!control!frequency!remained!36.4%!(n=44).!! A>C, Lateral, views, of, 3, days, old, larvae., Close! up! lateral! views! onto! anterior! are! shown! on! the! right.! Arrows! indicate! cardiac! oedema.! DBG,! Close! up! lateral! views! onto! cloaca! area! of! 3! dpf! zebrafish!larvae.!arrows!point!at!cloaca.!abbrevia7ons:!hg,!hindgut,!pd,!pronephric!duct.!