CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc CWC22_Hs CWC22_Mm CWC22_Gg CWC22_Xl CWC22_Dm CWC22_Ce CWC22_Sp CWC22_Sc 1 ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- MGESDAESDSSSNSSSSDTSSGSDSDARSESSSSESSGREEEEAKQEESAKDAKKTDDTDRGEKRAKERDAGQDEQPTEQKKTPAAEPRSERQHISHSAGVEKQQEEAVAAAEAESEKLNEAKKVETPVQRKEEAEASS ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- 1 1 1 140 1 1 ----------------------------------------------MKSSVAQIKPSSGHDRRENLNSYQ-RNSSPED------------------------------------------RYEE------QER-- SPRD - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MK S S V A HMK - SSGHNRRETHSSYR-RSSSPED------------------------------------------RYTE------QER-- SPRD ------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------MKSSVAQVR-GSNYDKVDTRSSSE-RSSSPEDSNEERSPSPRDRGYSNRSRDYSDRDRYEDRSRSGRYDRSDESRRRE------RERSTSPRD VTKELNSPKAQEENAARELEERRKDEEQPVTTNGSSKESPVEAAAETVKPPTAD-HIEEGEITDKD- EDDLPTKEEKKA--VASKSPPKETQRKQSRSPDGKRRRPRSSSRSPSPSSRRRRRSRSKGSRTRSRSKSPIR ------------------------------------------MSSSRSQSPE---TPKAASEEREEEEKESSEQPDTPKTSSEKSASRSQSPRESREVSQETETSENQEKIKEKDD---GDDQPGTPNSYRSRETSPAP -----------------------------------------------------------------------------------------------------------------------------------------ME ------------------------------------------------------------------------------------------------------------------------------------------- 43 42 1 86 275 92 3 1 RDYFDYSRSDYEHSRRGRSYDSS-----MESRNRDRE--KRRERERDTD-RKRSRKSP--- SPGRRNPET------- SVT---QSSSAQDEPATKKK----KDELDPLLTRTGGAYIPPAKLRMMQEQITD----KNSL RGYSDYSRSDYERSRRGYSYDDS-----MESRSRDRE--KRRERERDADHRKRSRKSP--- SPDR- S P A R - - - - - - - G G G - - - Q S S - P Q E E P T W K K K - - - - K D E L D P L L T R T G G A Y I P P A K L R MMQ E Q I T D - - - - K S S L ----------------------------ME-RGRDRYYEKWEERDYD---RRRKRRLS--- SPDHRSPER------- STV---QGSNTHEEATSKKK----KEEVDPILTRTGGAYIPPAKLRMMQEQITD----KNSL RGYTDRRRGYSDRDGYGNDRSRN-------GRYDRSEDNREREKRQNYQDRDYEKRSP---PARRRSPPA-------RRSEEQTEEQNQTEPPVKKK----KEELDPILTRTGGAYIPPARLRMMQEQITD----KSSM RRSNSLERRRVERQRRHEERDKRDEERAKEREKRHQKGEPTSSRRRDDSREKKRSPERKRDRSSSTPKSKSSKTPRHTETTETNADNETVTEPAAKITERQRKTVDVLTSRTGGAYIPPAKLRMMQSQITD----KSSA KR- SKETRESESPEKSP---------------------VRSRSPRRSSARSP-- S R S P R R R R E - - R S S E - - - - - - - - - - - - - - - - - R K Q S E E P A P L P E K K K K E P L D I L R T R T G G A Y I P P A K L R L MQ Q Q I S D - - - - K Q S E KEDKSFGIGMLDYNRENPESSGHSRVAVIRKQKTEQEN-------NNLSWEDRHYIPDLHKSNKIKTPTS----------------- LADEKKSHNELDPKAQIKKLMETRSGGTYIPPAKLKALQAQLTD----VNTP --------------------------------------------------------------------------------------------------------------------------------MSTATIQDEDI 153 151 87 200 410 184 114 12 AYQRMSWEALKKSINGLINKVNISNISI- I IQELLQENIVRGRGLLSRSVLQAQSASPI-- FTHVYAALVAI INSKFPQIGELILKRLILNFRKGYRRNDKQLCLTASKFVAHLINQNVAHEVLCLEMLTLLLERPTDD AYQRMSWEALKKSINGLINKVNISNISI- I IQELLQENIVRGRGLLSRSVLQAQSASPI-- FTHVYAALVAI INSKFPQIGELILKRLILNFRKGYRRNDKQLCLTASKFVAHLINQNVAHEVLCLEMLTLLLERPTDD AYQRMSWEALKKSINGLVNKVNVSNIEN- I IYELLQENIVRGRGLLSRSILQAQGASPI-- FTHVYAALVAI INSKFPNIGELILKRLILNFRKGYRRNDKQLCLTSSKFVAHLMNQNVAHEVLCLEMLTLLLERPTDD AYQRMSWEALKKSINGLVNKVNVSNIGN- I IQELLQENIVRGRGLLARSVLQAQSASPI-- FTHVYAALVSI INSKFPHIGELILKRLILNFRKGYRRNDKQLCLTSSKFVAHLINQNVAHEVLALEMLTLLLERPNDD AYQRIAWEALKKSIHGYINKVNVTNIAI- ITRELLRENIVRGRGLLSRSI IQAQAASPT-- FTHVYAALVSI INSKFPNIGELLLKRLVIQFRRAFRRNDKMVCMSATRFIGHLVNQRVAHEILALEILTLLVETPTDD QYQRMNWERMKKKIHGLVNRVNAKNLVQ- IVRELLQENVIRSKGLLCRDI IQAQAFSPG-- FSNVYAALAAVINSKFPHVGELLLRRLIVQFKRSFRRNDRGVTVNVIKFIAHLINQQVAHEVLALEIMILMLEEPTDD EYQRMQWEALKKSINGLINKVNKSNIRD- I IPELFQENI IRGRALYCRSIMKAQAASLP-- FTPIYAAMTAVINTKFPQIGELLLTRLIVQFRKSFQRNDKSMCISSSSFIAHLINQKIAHEIVGLQILAVLLERPTND KFQRENWEMIRSHVSPI ISNLTMD-NLQESHRDLFQVNILIGRNI ICKNVVDFTLNKQNGRLIPALSALIALLNSDIPDIGETLAKELMLMFVQQFNRKDYVSCGNILQCLSILFLYDVIHEIVILQILLLLLE----- 289 287 223 336 546 320 250 145 -- SVEVAIGFLKECGLKLTQVSPRGINAIFERLRN- ILHESE----- IDKRVQYMIEVMFAVRKDGFKD-HPI ILEGLDLVEEDDQFTHM- LPLEDDYNPEDVLNVFKM--DPNFMENEEKYKAIKKEILDEGDTDSNT -- SVEVAIGFLKECGLKLTQVSPRGINAIFERLRN- ILHESE----- IDKRVQYMIEVMFAVRKDGFKD-HPVILEGLDLVEEDDQFTHM- LPLEDDYNPEDVLNVFKM--DPNFMENEEKYKAIKKEILDEGDSDSNT -- SIEVAIGFLKESGLKLTEVSPRGINAIFDRLRH- ILHESK----- IDMRVQYMIEVMFAVRKDGFKD-HPI IPEGLDLVEEEDQFTHM- LPLEDEYNPEDVLNVFKM--DPNFMENEEKYKALKKEILDEGDSESEP -- SVEVAIGFLKESGLKLTQVTPRGINAIFERLRN- ILHESE----- IDKRVQYMIEVMFAVRKDGFKD-HPVIPEGLDLVEEEDQFTHM- LPLEDDYNQEDVLNVFKM--DPDFLENEEKYKAIKKEILDEGDSDSEG -- SVEVAIAFLKECGMKLTEVSSKGIGAIFEMLRN- ILHEGK----- LDKRVQYMIEVLFQIRKDGFKD-HQAVVPELELVEEDDQFTHL-MMLDEATETEDILNVFKF--DDNYAENEDKYKGLSREILGSDDGSS- S -- SVEVAIAFLKECGAKLLEIAPAALNSVYDRLRA- ILMETERSENALDRRIQYMIETAMQIRKDKFAA-YPAVIEDLDLIEEEDQI IHT- LNLEDAVDPENGLNVFKL--DPEFEKNEEVYEEIRKEI IGNADI---- -- SIEIAVMLLREIGAYLAEVSTRAYNGVFERFRTILHEGQLER------RTQFI IEVLFQTRKDKFKN-NPTIPQELDLVEEEDQITHY- ISLDDNLDVQESLGIFHY--DPDYEENEKKYDAIKHEILGEEDDD--- KNSLRLVIAVMKICGWKLALVSKKTHDMIWEKLRYILQTQELS------ STLRESLETLFEIRQKDYKSGSQGLFILDPTSYTVHTH-- SYIVSDED--- EANKELGNFEKCENFNELTMAFDTLRQKLLINNTSD--- 416 414 350 463 672 448 374 270 DQDAGSSEEDEE------ EEEEEGEEDEEGQKVTIHDKTEINLVSFRRTIYLAIQSSLDFEECAHKLLKMEFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCMLKKEYMESFEGIFKEQY-DTIHRLETNKLRNV DQGAGSSEDEEE----- EDEEEEGEDEEGGQKVTIHDKTEINLVSFRRTIYLAIQSSLDFEECAHKLLKMEFAESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCMLKKEYMESFESIFKEQY-DTIHRLETNKLRNV DQ E A G S S D E E E E - - - - - - - - - EDEEEDEDGQKVTVHDKTEINLVSFRRTIYLAIQSSLDFEECAHKLLKMDFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCMLKKEYMESFEAIFKEQY-DTIHRLETNKLRNV -DANEGSEDESE----- EEEEDGQEAGTEGEKMTIHDKTEVNLVAFRRTIYLAIQSSLDFEECAHKLIKMDFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCLLKKEYLEAFENIFKEQF- ETIHRLETNKLRNV GSGSGSDSDSDSDGESGSDAEK------KAEAGDI IDSTETNLIALRRTIYLTINSSLDYEECAHKLMKMQLKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCNINKIYIPPFEEIFKDTY-QTTHRLDTNRLRNV ----- SDEDGGDELDDEEEGSDVEEAPKK--TTEI IDNTDQNLTAFRREVYLTMQSSLDYQEAAHKLLKMKIPDSMQNELCAMLVDCCAQQRTYERFYGMLIERFCRLRLEYQQYFEKLCQDTY- STIHRIDITKLRNL ENEEDEEDSEETSESE- EDES------VNDEKPQVIDQTNASLVNLRKSIYLTIMSSVDFEECCHKLLKIDLPEGQEIELCNMVIECNSQERTYAKFYGLIGERFCKLSRTWRSTYEQCFKNYY- ETIHRYETNRLRNI -----TNEGSNSQ------------------- LQIYDMTSTNDVEFKKKIYLVLKSSLSGDEAAHKLLKLKIANNLKKSVVDI I I K S S L Q E S T F S K F Y S I L S E R M I T F H R S WQ T A Y N E T F E Q N Y T Q D I E D Y E T D Q L R I L 548 547 479 595 804 579 505 385 AKMFAHLLYTDSLPWSVLECIKLSEETTTSSSRIFVKIFFQELCEYMGLPKLNARLKDETLQPFFEGLLPRD-NPRNTRFAINFFTSIGLGGLTDELREHLKNTPKVIVAQKPDVEQ----------NKSSPSSSSSAS AKMFAHLLYTDSLPWSVLECIKLSEETTTSSSRIFVKIFFQELCEYMGLPKLNARLKDETLQPFFEGLLPRD-NPRNTRFAINFFTSIGLGGLTDELREHLKNTPKVIVAQKPEAEQ----------KKPALTSSSSES AKMFAHLLYTDSIPWSVLECI ILSEETTTSSSRIFVKIFFQELSEYMGLPNLNARLKDITLQPFFEGLLPRD-NPRNTRFAINFFTSIGLGGLTDELREHLKNAPKMIMTQKQDVES---------- SDSSSSSETDSS AKMFAHLLYTDSLPWSVLECMNLSEETTTSSSRIFVKIFFQELCEYMGLPKLNARLKDVTLQPFFQGLLPMD-NPKNTRFAINFFTSIGLGGLTDELREHLKNAPKMIMTQKQNVES---------- SDSSSDSSSGSE SKFFAHLLFTDAISWDVLECIQLNEDDTTSSSRIFIKILFQELAEYMGLGKLNAKLKDDVLVESIAGLFPKD-NPRNTRFSINFFTSIGLGGLTDDLRRFLKNAPKSVPAINAEILANAGGNPFRDGSAPAGNTKVAPS ARLIAHLLSTDAIDWKILADMKMTEEDTTSSGRIYIKYIFNELVEAMGMVKLHSRVTDPTLAHCFVGLFPRT-NPNSARFSINFFTMIGLGGLTLELREWLAKGLKKKKGMLDQL------------KAE- SSSDSSS- ALFFANLLSTDSIGWEVYDCVRLTEDDTTASSRIFLKIMFQEIVEALGLKSLVERLHDPNLVPYLHGLFPVD- EARNVRFSINYFTSIGLGALTEEMREYLLTMPKSEPKEQDSEGYS--------- SGSETGSTYSSS GKFWGHLISYEFLPMDCLKI IKLTEEESCPQGRIFIKFLFQELVNELGLDELQLRLNS---- SKLDGMFPLEGDAEHIRYSINFFTAIGLGLLTEDMRSRLTI IQ------ EVEDAEEEE----KKLREEEELEKLRKK 676 675 607 723 942 703 634 510 SSSESDSSD- SDSDSSDSS-- SESSSEESDSSSISS-HSSAS------ANDVRKKGHGKTRSKEVDKLIRNQQTNDRKQKERRQEHGHQET------RTERERRSEKHR--DQNSSG--- SNWRDPITKYTSDKDVPSE SSA- SDSSD- SESDSSESS-- SESSSDASDSSSSSSTQSSTSGITAHSAKGTRKKRQGKARGEEVDKLARGHQALERRREGGREDQRHQEG------RTERAR- SERRR--AQNSRD---ADWRDPLAKHIDDR--- SH SDSDSDSSS- SSSESSSSSDSSSSSSDSSDSDASKAKKRRTQKKNRESDKASRKKQEKKRKSLEKKTGRRRQEDRSDSESKSERNHRNLRE------AHRRDDTSKYHHRDESNGRDDYEAYRRDDVSKYHQRDESNGR SSSDSDSS------- SSSSS-- ESSSSSSDSDS--------------- SRRKKHSQKKKKSREAHKAASKKQAPDDRRKEAPKHKHQKEKY------DDKQSRKSKRDSKN---------------------------- SSSSSSSSSDTDSED---------- SSEEDSSSDSSSESSSSDSSSEPKKKRKRKDKDKKKSKKATKEKSKKTKNK--KKK-------------KKAEKEQ--------------------- EKEKEKQRKSKKEKEKD SSDSSDSSDSSDSDDS------ SDSSSDSSSSSESEPEPPKKKKKKNSEESSKKKEKENIGRRD-RGDKRAERHRDQSVENKDKD------------RRRRQDSDENRRPERGDDRKDRSKDRR----RQDSDDEDRKG YSSTYSRGRSYSRSTRSYSKSRSYSRSRSVTPINNINHKKYIRKDRELSPRGRERSSNRNSYSDLSRSSSLSRGRSRSYTPEGRLIESEDKGYRSRSSSPASRKYRSRQRYRRSYAGSTSRGRSFSRSPSYRRRLSMSC ARESQPTQGPKIHESRLFLQKDTRENSRSRSPFTVETR-KRARSRTPPRGSRNHRNRS--------------RTPPARRQRHR-------------------------------------------------------- 794 795 739 1035 819 773 RNNYSRVANDRD--QEMHIDLENKHGDPKKKRGERRNSFSENEKHTHRIKDSEN- FRRKDRSKSKEMNRKHSGSRS-DED-RYQNGAERRWEKSSRYSEQSRESKKNQDRRREKSPAKQK------------------- ENSHSRVGNGRE--QGSHREPEDRHGEPKKR-RERRDSFSENEKQRSRNQDSDN-VRRKDRSKSRERSRRHSGHKG-DDA-RCQNSAERRWEKPGRRPEQSRESKRSQDRRREKSPTTQK------------------- DNYHS--GRDRD--HERSKDLENKHSNSKLKKAERRASFSDDESYRHGSRDNGHRSRKRERSKSGERSYNKSSPREEEDDHRYRNGSERLREKYNHYSDQYRESRKHEDRRREYSPHRRK------------------- ------------------------------------------------------------------------------------------------------------------------------------------- KK--RKKEE------KKAAKKKSKHRRKSQESSDSSGSEDSDKSTSESSDSSNSSSDESDAEPQAKIKRQEHVEKN-KFRGRTQDSDEFNLEGPGSK--NRFQ-PNGNGQRRRDNSTGRERNRENSSYDRERNRGNSSY --RERREDS------------------------------GERR-RGDRDRRDRNKDQEDHREDRRDRSKDREDRRDRRRHDSDDDRKTRRDRSEERGGRRREVESDDRRRRR--------------------------- SVSYSRSPSPAARPISRSP-ARNKRSYDSLSYNRQYSPKVSRKRSNESRSPSPYTLRKQKTYHLNKRNEDFVVKMDKKGSESPVILAPWLREVDDGELYKDRNSESDSVRKQPRAD----------------------- ------------------------------------------------------------------------------------------------------------------------------------------- 1162 ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- DRERKRGNSSYDRERNRESSYDKERKNRNAVAHDRQRKRDRSRSYERPTIRENSAPREKRMESSRSEKDSRRGDRSSRNERSDRGERSDRGERSDRGERSDRGERSDRGERSDRGERSDREKERSRAKERERDRDRDLK ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- 1301 ------------------------------ ------------------------------ ------------------------------ ------------------------------ GQRERKRERDDGSRDRSRRERSSRRSKGRS ------------------------------ ------------------------------ ------------------------------ Supplementary Figure 1 Sequence conservation of CWC22 Sequence alignment of NCM from H. sapiens (Hs), M. musculus (Mm), G. gallus (Gg), X. laevis, (Xl), D. melanogaster (Dm), C. elegans (Ce), S. pombe (S.p.) and S. cerevisiae (Sc). Residues conserved between all the species are shaded in dark blue while residues in only some species are gradually shaded in lighter blue. The position of the domains MIF4G and MA3 are indicated on the top of the sequence with red and green lines, respectively. Nature Structural & Molecular Biology: doi:10.1038/nsmb.2380
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43421
862759231
R D Y F D Y S R S D Y E H S R R G R S Y D S S - - - - - M E S R N R D R E - - K R R E R E R D T D - R K R S R K S P - - - S P G R R N P E T - - - - - - - S V T - - - Q S S S A Q D E P A T K K K - - - - K D E L D P L L T R T G G A Y I P P A K L R MMQ E Q I T D - - - - K N S LR G Y S D Y S R S D Y E R S R R G Y S Y D D S - - - - - M E S R S R D R E - - K R R E R E R D A D H R K R S R K S P - - - S P D R - S P A R - - - - - - - G G G - - - Q S S - P Q E E P T WK K K - - - - K D E L D P L L T R T G G A Y I P P A K L R MMQ E Q I T D - - - - K S S L- - - - - - - - - - - - - - - - - - - - - - - - - - - - M E - R G R D R Y Y E K W E E R D Y D - - - R R R K R R L S - - - S P D H R S P E R - - - - - - - S T V - - - Q G S N T H E E A T S K K K - - - - K E E V D P I L T R T G G A Y I P P A K L R MMQ E Q I T D - - - - K N S LR G Y T D R R R G Y S D R D G Y G N D R S R N - - - - - - - G R Y D R S E D N R E R E K R Q N Y Q D R D Y E K R S P - - - P A R R R S P P A - - - - - - - R R S E E Q T E E Q NQ T E P P V K K K - - - - K E E L D P I L T R T G G A Y I P P A R L R MMQ E Q I T D - - - - K S S MR R S N S L E R R R V E R Q R R H E E R D K R D E E R A K E R E K R HQ K G E P T S S R R R D D S R E K K R S P E R K R D R S S S T P K S K S S K T P R H T E T T E T N A D N E T V T E P A A K I T E R Q R K T V D V L T S R T G G A Y I P P A K L R MMQ S Q I T D - - - - K S S AK R - S K E T R E S E S P E K S P - - - - - - - - - - - - - - - - - - - - - V R S R S P R R S S A R S P - - S R S P R R R R E - - R S S E - - - - - - - - - - - - - - - - - R K Q S E E P A P L P E K K K K E P L D I L R T R T G G A Y I P P A K L R L MQ Q Q I S D - - - - K Q S EK E D K S F G I GM L D Y N R E N P E S S G H S R V A V I R K Q K T E Q E N - - - - - - - N N L S W E D R H Y I P D L H K S N K I K T P T S - - - - - - - - - - - - - - - - - L A D E K K S H N E L D P K AQ I K K L M E T R S G G T Y I P P A K L K A L Q AQ L T D - - - - V N T P- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M S T A T I Q D E D I
15315187
20041018411412
A Y Q R M SW E A L K K S I N G L I N K V N I S N I S I - I I Q E L L Q E N I V R G R G L L S R S V L Q AQ S A S P I - - F T H V Y A A L V A I I N S K F P Q I G E L I L K R L I L N F R K G Y R R N D K Q L C L T A S K F V A H L I NQ N V A H E V L C L E M L T L L L E R P T D DA Y Q R M SW E A L K K S I N G L I N K V N I S N I S I - I I Q E L L Q E N I V R G R G L L S R S V L Q AQ S A S P I - - F T H V Y A A L V A I I N S K F P Q I G E L I L K R L I L N F R K G Y R R N D K Q L C L T A S K F V A H L I NQ N V A H E V L C L E M L T L L L E R P T D DA Y Q R M SW E A L K K S I N G L V N K V N V S N I E N - I I Y E L L Q E N I V R G R G L L S R S I L Q AQ G A S P I - - F T H V Y A A L V A I I N S K F P N I G E L I L K R L I L N F R K G Y R R N D K Q L C L T S S K F V A H L MNQ N V A H E V L C L E M L T L L L E R P T D DA Y Q R M SW E A L K K S I N G L V N K V N V S N I G N - I I Q E L L Q E N I V R G R G L L A R S V L Q AQ S A S P I - - F T H V Y A A L V S I I N S K F P H I G E L I L K R L I L N F R K G Y R R N D K Q L C L T S S K F V A H L I NQ N V A H E V L A L E M L T L L L E R P N D DA Y Q R I AW E A L K K S I H G Y I N K V N V T N I A I - I T R E L L R E N I V R G R G L L S R S I I Q A Q A A S P T - - F T H V Y A A L V S I I N S K F P N I G E L L L K R L V I Q F R R A F R R N D K M V C M S A T R F I G H L V NQ R V A H E I L A L E I L T L L V E T P T D DQ Y Q R MNW E R M K K K I H G L V N R V N A K N L V Q - I V R E L L Q E N V I R S K G L L C R D I I Q A Q A F S P G - - F S N V Y A A L A A V I N S K F P H V G E L L L R R L I V Q F K R S F R R N D R G V T V N V I K F I A H L I NQ Q V A H E V L A L E I M I L M L E E P T D DE Y Q R MQW E A L K K S I N G L I N K V N K S N I R D - I I P E L F Q E N I I R G R A L Y C R S I M K AQ A A S L P - - F T P I Y A AM T A V I N T K F P Q I G E L L L T R L I V Q F R K S F Q R N D K S M C I S S S S F I A H L I NQ K I A H E I V G L Q I L A V L L E R P T N DK F Q R E NW EM I R S H V S P I I S N L T MD - N L Q E S H R D L F Q V N I L I G R N I I C K N V V D F T L N K Q N G R L I P A L S A L I A L L N S D I P D I G E T L A K E L M L M F V Q Q F N R K D Y V S C G N I L Q C L S I L F L Y D V I H E I V I L Q I L L L L L E - - - - -
289287223336546320250145
- - S V E V A I G F L K E C G L K L T Q V S P R G I N A I F E R L R N - I L H E S E - - - - - I D K R V Q Y M I E V M F A V R K D G F K D - H P I I L E G L D L V E E D DQ F T HM - L P L E D D Y N P E D V L N V F K M - - D P N F M E N E E K Y K A I K K E I L D E G D T D S N T- - S V E V A I G F L K E C G L K L T Q V S P R G I N A I F E R L R N - I L H E S E - - - - - I D K R V Q Y M I E V M F A V R K D G F K D - H P V I L E G L D L V E E D DQ F T HM - L P L E D D Y N P E D V L N V F K M - - D P N F M E N E E K Y K A I K K E I L D E G D S D S N T- - S I E V A I G F L K E S G L K L T E V S P R G I N A I F D R L R H - I L H E S K - - - - - I DM R V Q Y M I E V M F A V R K D G F K D - H P I I P E G L D L V E E E DQ F T HM - L P L E D E Y N P E D V L N V F K M - - D P N F M E N E E K Y K A L K K E I L D E G D S E S E P- - S V E V A I G F L K E S G L K L T Q V T P R G I N A I F E R L R N - I L H E S E - - - - - I D K R V Q Y M I E V M F A V R K D G F K D - H P V I P E G L D L V E E E DQ F T HM - L P L E D D Y NQ E D V L N V F K M - - D P D F L E N E E K Y K A I K K E I L D E G D S D S E G- - S V E V A I A F L K E C GM K L T E V S S K G I G A I F E M L R N - I L H E G K - - - - - L D K R V Q Y M I E V L F Q I R K D G F K D - HQ A V V P E L E L V E E D DQ F T H L - MM L D E A T E T E D I L N V F K F - - D D N Y A E N E D K Y K G L S R E I L G S D D G S S - S- - S V E V A I A F L K E C G A K L L E I A P A A L N S V Y D R L R A - I L M E T E R S E N A L D R R I Q Y M I E T AMQ I R K D K F A A - Y P A V I E D L D L I E E E DQ I I H T - L N L E D A V D P E N G L N V F K L - - D P E F E K N E E V Y E E I R K E I I G N A D I - - - -- - S I E I A V M L L R E I G A Y L A E V S T R A Y N G V F E R F R T I L H E GQ L E R - - - - - - R T Q F I I E V L F Q T R K D K F K N - N P T I P Q E L D L V E E E DQ I T H Y - I S L D D N L D V Q E S L G I F H Y - - D P D Y E E N E K K Y D A I K H E I L G E E D D D - - -K N S L R L V I A V M K I C GWK L A L V S K K T H DM I W E K L R Y I L Q T Q E L S - - - - - - S T L R E S L E T L F E I R Q K D Y K S G S Q G L F I L D P T S Y T V H T H - - S Y I V S D E D - - - E A N K E L G N F E K C E N F N E L T M A F D T L R Q K L L I N N T S D - - -
416414350463672448374270
DQ D A G S S E E D E E - - - - - - E E E E E G E E D E E GQ K V T I H D K T E I N L V S F R R T I Y L A I Q S S L D F E E C A H K L L K M E F P E S Q T K E L C NM I L D C C AQ Q R T Y E K F F G L L A G R F C M L K K E Y M E S F E G I F K E Q Y - D T I H R L E T N K L R N VDQ G A G S S E D E E E - - - - - E D E E E E G E D E E G GQ K V T I H D K T E I N L V S F R R T I Y L A I Q S S L D F E E C A H K L L K M E F A E S Q T K E L C NM I L D C C AQ Q R T Y E K F F G L L A G R F C M L K K E Y M E S F E S I F K E Q Y - D T I H R L E T N K L R N VDQ E A G S S D E E E E - - - - - - - - - E D E E E D E D GQ K V T V H D K T E I N L V S F R R T I Y L A I Q S S L D F E E C A H K L L K MD F P E S Q T K E L C NM I L D C C AQ Q R T Y E K F F G L L A G R F C M L K K E Y M E S F E A I F K E Q Y - D T I H R L E T N K L R N V- D A N E G S E D E S E - - - - - E E E E D GQ E A G T E G E K M T I H D K T E V N L V A F R R T I Y L A I Q S S L D F E E C A H K L I K M D F P E S Q T K E L C NM I L D C C AQ Q R T Y E K F F G L L A G R F C L L K K E Y L E A F E N I F K E Q F - E T I H R L E T N K L R N VG S G S G S D S D S D S D G E S G S D A E K - - - - - - K A E A G D I I D S T E T N L I A L R R T I Y L T I N S S L D Y E E C A H K L M K MQ L K P GQ E I E L C HM F L D C C A E Q R T Y E K F Y G L L A Q R F C N I N K I Y I P P F E E I F K D T Y - Q T T H R L D T N R L R N V- - - - - S D E D G G D E L D D E E E G S D V E E A P K K - - T T E I I D N T DQ N L T A F R R E V Y L T MQ S S L D Y Q E A A H K L L K M K I P D S MQ N E L C AM L V D C C AQ Q R T Y E R F Y GM L I E R F C R L R L E Y Q Q Y F E K L C Q D T Y - S T I H R I D I T K L R N LE N E E D E E D S E E T S E S E - E D E S - - - - - - V N D E K P Q V I DQ T N A S L V N L R K S I Y L T I M S S V D F E E C C H K L L K I D L P E GQ E I E L C NM V I E C N S Q E R T Y A K F Y G L I G E R F C K L S R T WR S T Y E Q C F K N Y Y - E T I H R Y E T N R L R N I- - - - - T N E G S N S Q - - - - - - - - - - - - - - - - - - - L Q I Y DM T S T N D V E F K K K I Y L V L K S S L S G D E A A H K L L K L K I A N N L K K S V V D I I I K S S L Q E S T F S K F Y S I L S E R M I T F H R S WQ T A Y N E T F E Q N Y T Q D I E D Y E T DQ L R I L
548547479595804579505385
A K M F A H L L Y T D S L P W S V L E C I K L S E E T T T S S S R I F V K I F F Q E L C E Y MG L P K L N A R L K D E T L Q P F F E G L L P R D - N P R N T R F A I N F F T S I G L G G L T D E L R E H L K N T P K V I V A Q K P D V E Q - - - - - - - - - - N K S S P S S S S S A SA K M F A H L L Y T D S L P W S V L E C I K L S E E T T T S S S R I F V K I F F Q E L C E Y MG L P K L N A R L K D E T L Q P F F E G L L P R D - N P R N T R F A I N F F T S I G L G G L T D E L R E H L K N T P K V I V A Q K P E A E Q - - - - - - - - - - K K P A L T S S S S E SA K M F A H L L Y T D S I P W S V L E C I I L S E E T T T S S S R I F V K I F F Q E L S E Y MG L P N L N A R L K D I T L Q P F F E G L L P R D - N P R N T R F A I N F F T S I G L G G L T D E L R E H L K N A P K M I M T Q K Q D V E S - - - - - - - - - - S D S S S S S E T D S SA K M F A H L L Y T D S L P W S V L E C MN L S E E T T T S S S R I F V K I F F Q E L C E Y MG L P K L N A R L K D V T L Q P F F Q G L L P MD - N P K N T R F A I N F F T S I G L G G L T D E L R E H L K N A P K M I M T Q K Q N V E S - - - - - - - - - - S D S S S D S S S G S ES K F F A H L L F T D A I S WD V L E C I Q L N E D D T T S S S R I F I K I L F Q E L A E Y MG L G K L N A K L K D D V L V E S I A G L F P K D - N P R N T R F S I N F F T S I G L G G L T D D L R R F L K N A P K S V P A I N A E I L A N A G G N P F R D G S A P A G N T K V A P SA R L I A H L L S T D A I DWK I L A DM K M T E E D T T S S G R I Y I K Y I F N E L V E AMGM V K L H S R V T D P T L A H C F V G L F P R T - N P N S A R F S I N F F T M I G L G G L T L E L R E W L A K G L K K K K GM L DQ L - - - - - - - - - - - - K A E - S S S D S S S -A L F F A N L L S T D S I GW E V Y D C V R L T E D D T T A S S R I F L K I M F Q E I V E A L G L K S L V E R L H D P N L V P Y L H G L F P V D - E A R N V R F S I N Y F T S I G L G A L T E E M R E Y L L T M P K S E P K E Q D S E G Y S - - - - - - - - - S G S E T G S T Y S S SG K F WG H L I S Y E F L P MD C L K I I K L T E E E S C P Q G R I F I K F L F Q E L V N E L G L D E L Q L R L N S - - - - S K L D GM F P L E G D A E H I R Y S I N F F T A I G L G L L T E DM R S R L T I I Q - - - - - - E V E D A E E E E - - - - K K L R E E E E L E K L R K K
676675607723942703634510
S S S E S D S S D - S D S D S S D S S - - S E S S S E E S D S S S I S S - H S S A S - - - - - - A N D V R K K G H G K T R S K E V D K L I R NQ Q T N D R K Q K E R R Q E H G HQ E T - - - - - - R T E R E R R S E K H R - - DQ N S S G - - - S NWR D P I T K Y T S D K D V P S ES S A - S D S S D - S E S D S S E S S - - S E S S S D A S D S S S S S S T Q S S T S G I T A H S A K G T R K K R Q G K A R G E E V D K L A R G HQ A L E R R R E G G R E DQ R HQ E G - - - - - - R T E R A R - S E R R R - - A Q N S R D - - - A DWR D P L A K H I D D R - - - S HS D S D S D S S S - S S S E S S S S S D S S S S S S D S S D S D A S K A K K R R T Q K K N R E S D K A S R K K Q E K K R K S L E K K T G R R R Q E D R S D S E S K S E R N H R N L R E - - - - - - A H R R D D T S K Y H H R D E S N G R D D Y E A Y R R D D V S K Y HQ R D E S N G RS S S D S D S S - - - - - - - S S S S S - - E S S S S S S D S D S - - - - - - - - - - - - - - - S R R K K H S Q K K K K S R E A H K A A S K K Q A P D D R R K E A P K H K HQ K E K Y - - - - - - D D K Q S R K S K R D S K N - - - - - - - - - - - - - - - - - - - - - - - - - - - -S S S S S S S S S D T D S E D - - - - - - - - - - S S E E D S S S D S S S E S S S S D S S S E P K K K R K R K D K D K K K S K K A T K E K S K K T K N K - - K K K - - - - - - - - - - - - - K K A E K E Q - - - - - - - - - - - - - - - - - - - - - E K E K E K Q R K S K K E K E K DS S D S S D S S D S S D S D D S - - - - - - S D S S S D S S S S S E S E P E P P K K K K K K N S E E S S K K K E K E N I G R R D - R G D K R A E R H R DQ S V E N K D K D - - - - - - - - - - - - R R R R Q D S D E N R R P E R G D D R K D R S K D R R - - - - R Q D S D D E D R K GY S S T Y S R G R S Y S R S T R S Y S K S R S Y S R S R S V T P I N N I N H K K Y I R K D R E L S P R G R E R S S N R N S Y S D L S R S S S L S R G R S R S Y T P E G R L I E S E D K G Y R S R S S S P A S R K Y R S R Q R Y R R S Y A G S T S R G R S F S R S P S Y R R R L S M S CA R E S Q P T Q G P K I H E S R L F L Q K D T R E N S R S R S P F T V E T R - K R A R S R T P P R G S R N H R N R S - - - - - - - - - - - - - - R T P P A R R Q R H R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
794795739
1035819773
R N N Y S R V A N D R D - - Q E MH I D L E N K H G D P K K K R G E R R N S F S E N E K H T H R I K D S E N - F R R K D R S K S K E MN R K H S G S R S - D E D - R Y Q N G A E R R W E K S S R Y S E Q S R E S K K NQ D R R R E K S P A K Q K - - - - - - - - - - - - - - - - - - -E N S H S R V G N G R E - - Q G S H R E P E D R H G E P K K R - R E R R D S F S E N E K Q R S R NQ D S D N - V R R K D R S K S R E R S R R H S G H K G - D D A - R C Q N S A E R R W E K P G R R P E Q S R E S K R S Q D R R R E K S P T T Q K - - - - - - - - - - - - - - - - - - -D N Y H S - - G R D R D - - H E R S K D L E N K H S N S K L K K A E R R A S F S D D E S Y R H G S R D N G H R S R K R E R S K S G E R S Y N K S S P R E E E D D H R Y R N G S E R L R E K Y N H Y S DQ Y R E S R K H E D R R R E Y S P H R R K - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -K K - - R K K E E - - - - - - K K A A K K K S K H R R K S Q E S S D S S G S E D S D K S T S E S S D S S N S S S D E S D A E P Q A K I K R Q E H V E K N - K F R G R T Q D S D E F N L E G P G S K - - N R F Q - P N G N GQ R R R D N S T G R E R N R E N S S Y D R E R N R G N S S Y- - R E R R E D S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G E R R - R G D R D R R D R N K DQ E D H R E D R R D R S K D R E D R R D R R R H D S D D D R K T R R D R S E E R G G R R R E V E S D D R R R R R - - - - - - - - - - - - - - - - - - - - - - - - - - -S V S Y S R S P S P A A R P I S R S P - A R N K R S Y D S L S Y N R Q Y S P K V S R K R S N E S R S P S P Y T L R K Q K T Y H L N K R N E D F V V K MD K K G S E S P V I L A P W L R E V D D G E L Y K D R N S E S D S V R K Q P R A D - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
1162
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Supplementary Figure 1 Sequence conservation of CWC22
Sequence alignment of NCM from H. sapiens (Hs), M. musculus (Mm), G. gallus (Gg), X. laevis, (Xl), D. melanogaster (Dm), C. elegans (Ce), S. pombe (S.p.) and S. cerevisiae (Sc). Residues conserved between all the species are shaded in dark blue while residues in only some species are gradually shaded in lighter blue. The position of the domains MIF4G and MA3 are indicated on the top of the sequence with red and green lines, respectively.
Supplementary Figure 2 Western blotting with anti-CWC22 antibodies
Whole cell lysates from HeLa and HEK293T cells were loaded onto a 10% SDS-PAGE, transferred onto nitrocellulose membrane before western blotting with purified polyclonal anti-CWC22 antibodies raised against the N- or C-terminal extremity of human CWC22 (anti-CWC22n and anti-CWC22c, respectively). The position of CWC22 is indicated.
Supplementary Figure 3 Localization of CWC22 and EJC core proteins in vivo
(a) HeLa cells transfected with GFP-CWC22 (a-h) were fixed and stained using anti-SC35 (a), or anti-eIF4AIII (e) and counterstained with dapi (blue). Merged images are shown on the right panels. (b) Untransfected HeLa cells were fixed and stained using anti-SC35 (a) and anti-CWC22 (b) and counterstained with dapi (blue). (c) HeLa cells were co-transfected with GFP-CWC22 and Cherry-MAGOH (a-d), Cherry-MLN51-SELOR (e-h) or Cherry-Y14 (i-l) and counterstained with dapi (blue). Merged images are shown on the right panels.
Supplementary Figure 4 Affinity purification of recombinant CWC22-S
(a) Purification of 6xhistidine and CBP-tagged CWC22-S protein from E. coli using gravity-flow chromatography. Fractions were collected from each of the purification steps and assayed on a 12% SDS-PAGE and Coomassie staining. S: soluble lysate fraction; P: pellet corresponding to the insoluble lysate fraction; FTn: flow-through of the Ni-NTA column; W: high salt wash of the Ni-NTA column; En: elution of the Ni-NTA column; FTc: flow-through of the calmodulin column; Ec: Elution of the calmodulin column. M, protein molecular weight standards (kD). (b) Same as a except that the heterodimer CBP-eIF4AIII/CWC22-S-His was purified.
Supplementary Figure 5 Effect of siRNAs on splicing of selected introns
(a) Total RNAs from HEK293 cells transfected with siRNAs specific to GFP, Y14, CWC22 (two different siRNAs were employed, siRNA CWC22-1 and CWC22-2) and Upf1 were reverse-transcribed. Olignucleotides complementary to MAT2A exons 7 and 8 were used for PCR amplification. Genomic DNA (gDNA) was used as a control. Position of products corresponding to pre-mRNA and mRNA are indicated at right. (b) Same as a except that oligonucleotides complementary to MAT2A exons 3 and 4 were used for PCR amplification. (c) Same as a except that oligonucleotides complementary to GAPDH exons 5 and 6 were used for PCR amplification.
For immunofluorescence, HeLa cells at 50-70% confluency were plated on 18mm glass
coverslips and transfected with 2 µg of various plasmids using JetPEITM transfection reagent
(Polyplus transfection, Ozyme) following manufacturer’s instructions. After 24 h, cells were
washed with PBS, fixed for 15 min at room temperature in 4% paraformaldehyde in PBS, and
permeabilized for 3 min with 2.5% Triton X-100 and 5 min with 0.5% Triton X-100 in PBS.
After blocking in 5% bovine serum albumin in PBS, cells were incubated at room temperature
with the primary antibodies rabbit anti-eIF4AIII or mouse anti-SC35 for 1h. Cells were
washed twice in PBS and incubated for 1 h with Cy3 secondary antibody (1:1000). Cells
were washed three times in PBS, and nuclei were stained with DAPI. Slides were mounted
and observed with a fluorescence microscope (NikonTi). For S2 cells, stable cell lines
expressing DmCWC22-RFP were done as previously described1. Transient transfection
DmMAGO-GFP, DmY14-GFP and DmeIF4AIII-GFP were done with Effectene according to
the manufacturer (Qiagen). Transfected cells lines were fixed with 4% para-fomaldehyde and
DAPI stained (Invitrogen) according to standard protocol. Imaging was done on an Axiovert
206 inverted bright field microscope (Zeiss).
Primers for RT-qPCR (Supplementary Table 2)
For MAT2A, Forward: HLH1561; Reverse: HLH1562
For GAPDH, Forward: HLH1567; Reverse: HLH1568
For FUSIP, Forward: HLH1569; Reverse: HLH1570
Splicing control of endogenous transcripts
To check splicing, 1µL of cDNA or 250ng of genomic DNA were amplified by PCR with 1u of
Dreamtaq Green (Fermentas), 0,1mM of each dNTP, 0.1µM of each oligonucleotide
(HLH1820 & 1821 for exons 7-8 of MAT2A; HLH1816 & 1817 for exons 3-4 of MAT2A;
HLH1824 & 1825 for exons 5-6 of GAPDH, Supplementary Table 2) in a 20µL volume
reaction. PCR products were loaded in a 2% agarose gel with BET staining.
References 1. Florens, L. & Washburn, M.P. Proteomic analysis by multidimensional protein identification technology. Methods Mol Biol 328, 159-75 (2006). 2. Paoletti, A.C. et al. Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc Natl Acad Sci U S A 103, 18928-33 (2006). 3. Saulière, J. et al. The exon junction complex differentially marks spliced junctions. Nat Struct Mol Biol 17, 1269-71 (2010).
