Cloning and expression analysis of MBLL cDNA

Cloning and expression analysis of MBLL cDNADU Guangwei, ZHOU Yan, CHEN Jianhe, WANG Junhua, YIN Bin,YUAN Jiangang & QIANG BoqinNational Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences and Chinese Academy of MedicalSciences, Peking Union Medical College, National Center of Human Oenome Research, Bejjing 100005, ChinaCorrespondence should be addressed to Yuan Jiangang (e-mail: yuanjiangang@ yahoo.com)Abstract The mb/ (muscleblind) gene of Drosophila encodes a nuclear protein which contains twoCysgHis motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila'sphotoreceptors. Primers have been designed according to human EST086139, which is highlyhomologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNAclone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains anopen reading frame which encodes 255 amino acids and has 4 CysgHis motifs (GenBank Acc.AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northernblot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLLis a widely expressed gene, but the expression amounts diffr in these tissues.Keywords: MBLL, Cys,His zinc finger motif , cDNA cloning, tssue expression.Many development procedures in vertebrate and some invertebrate organisms are conservativel!.As an important model organism, Drosophila provides lots of information for the elucidation ofvertebrate' s developmental mechanism. Many genes related to human development have been clonedaccording to the information of Drosophila' s development related genes. Banfi et al.l21 have analyzed allknown genes in Drosophila database- Flybase. Through human EST database retrieval, 66 humancDNAs that are significantly homologous to Drosophila counterparts are found and localized in humanchromosomes, which provided information for cloning of human corresponding genes.The mbl (muscleblind) of Drosophila encodes a nuclear protein which contains two CyszHismotifs. The mutation of mbl disturbs the differentiation of all its photoreceptors')!. Mbl can also inhibitthe eye development anomaly caused by activation of Jun (sE-JunASP). Begemann et al.531 regardedMbl as a general factor for photoreceptor differentiation. Until now, the knowledge of molecularmechanism for human eye development is poor. For understanding the function of human mblhomologue, PCR primers for cDNA library screening were designed according to EST AA086139. Ahuman cDNA clone MBLL ( muscleblind-like) was obtained. MBLL cDNA was 2595 bp and its openreading frame encodes a protein that has 255 amino acids and contains 4 Cys;His zinc finger motifs.Tissue expression analysis showed that MBLL is widely expressed.1 Material and method( i ) Screening of arrayed human fetal brain cDNA library.Primers 15456, 15457 were designedaccording to EST AA086139; and primers 15454, 15455 were designed according to EST 13608.Arrayed human fetal brain cDNA library was screened by PCR method4. The reaction condition is94C pre-denaturing for 3 min, 94°C denaturing for 30 s, 55"C annealing for 30 s, 72C elongatingfor 40 s, 35 cycles, 72C for 10 min. Every positive tube contained about 1 200 independent clones.Single clone was isolated from positive tubes through routine hybridization method's. Positive ADR2phage clones were isolated and converted to pDR2 plasmid by introducing入DR2 phages into AM1 hostcells as described in Clontech's manual. Plasmid DNA was isolated by conventional alkali lysis andPEG precipitation. DNA sequencing was performed according to the standard method on ABI 377autosequencer. The full length cDNA sequence was廿中国煤化工10(ii ) Sequence analysis. Hydrophilic analysisHOPP.THR program ofPROSIS software. The cDNA sequence and predicteYHC N M H Grched against GenBankusing Gapped BLAST program!6l (website: http://www.ncbi.nlm.nih.gov). Protein motifs search andmultiple sequences alignment were carried out by using Prosite databasel]l and CLUSTAL W programat BCM search browserl8).Chinese_ Science BulletinVol. 45 No. 1January 200011CTQAAGOTAAAATTrTcCAGATACaoCAGACGGCTTTCAGAGTACAATAAACAGGGAT5960 anG AC TAT TT入CAT GGA AGT TTC rTr CTC ATG ATG Coo ToG AGA AGC CTC GQC CAC TTG 11920 GTT CTG CCA GAT OTT ccT GGG GTT ACT GTA AAt GGG AAG QAc A0G cao AGC TAA ACA A00.179180 rTT ArC ATT Taa MAG rac CTG YTG TGA AGT CAC TTT TeC rGG A入A ACT GCA GCT rOO AOC239240 rr crr ror Arr cAc ATC, CCA cTC rTC TGT CAA GTA CAC TTT ACC CTG ACc TTA TGA oTG 299300 aAT QAA GAT ACC TCA OTT GTC TGA CTT TaC CAA TTG CTT AAT TTC AGA ATT TAA AAA G0G359360 GA AGA AA ACA TCC TaC TAA AAT ATG AAC ATC TGA GTG TCT TAT TTT CCA ACA TCG TCA419420 ATA acT GTG AGC GTC AGC ATT AA TAT TCT ccc AAG OAG TaC CAT GAT ATT GAA GTC ACT479480 TTA TA ATA ACA acT ora TCT aCA AAA CAG TCA AGA QAC TCG GAC GTT GAA AsC CAo AGA 539540 TaA CAC TOA CA Tac TTT TAT rgc GgC CTA CCA TCT TTA AOT 00G ACA TAT ToA TTG ATa599600 AOT QAT rac CTG TCc ATA CAC TCT CTC ATC ATC CTG TTC CTT GGA TTO GAC TTC ACT AAG659660 CA TTT ATC ACT CAC CTT CAG ACT TAC ATG TOQ OAG TTT TCA CAA CAG TAG TTT TGG AAT 719720 CAT TAG AAC TTG GAT TQA TT CAT CAT TTA ACA GAA ACA AAC AGC CCA AAT TAC TTT ATC779780 ACC ATG GCT TTG AAC GTT acc CCA GTC AGA GAT ACA AA Too CTG ACA TTA GAA orC TOC83919日40 AGA CAG TTT CAA AGA GGA ACA TGC TCA CaC TCT. GAT GAA GAA TGC AAA TTT GCT CAT CCC 899203Cys,His motif 100 ccc AAA AOT TGT CAG GTT GAA AAT GGA AGA GTA ATT GCC ToC TTT GAT TCc CTA AAG GOC 95940 PKscQV .ENG RV_IACPDS_LKG 5960 CoT TOT TCO AGA GAG AAC TaC AAG TAT CTT CAC CCT CCG ACA CAC TTA AAA ACT CAA CTA 101s0_R_C___s__R_E___NC__KYI____HPPTHLKTQL7sCys,His motif21020 oAl ATT AAT GGA AGG AAC AT TTG ATT CAG CAA MMA act GCA GCA GCA ATG CTT acc CAG 10791080 CAG ATG CAA TT ATG TTT CCA GGA ACA CCA CTT CAT CCA GTG Ccc ACT TTC CCT GTA OGT 119!1001191140 ccc aCG ATA GGG ACA AAT ACG oCT ATT AGC TTT GCT CCT TAC CTA GCA CCT OTA ACC CCT 1199120PAIGTNTAIs.EAPYL.APVTP1391200 GGA GTT GQG TTG GTC CCA ACG GAA ATT CTG ccc ACC ACG ccT GTT ATT GTT CCC GGA AGT 12591401591260 ccA ccG orc ACT GTC ccG agc TCA ACT GCA ACT CAG AMA CTT CTC AGG ACT GAC AAA CTG 1319160PPvrVPGsTA里日KLLR.TDKL1791320 aAG GTA TGC AGG GAG TTC CAG CGA GGA AAc TOT GCC CGG GGA GAG ACC GAC TGC CaC 13791. C__RBP_RG _199CysHis motif 31380 GCA CAC ccc GCA GAC AGC ACC ATG ATC GAC ACA AGT GAC AAC ACC GTA ACC GTT TGT ATG 14392001440 GAT TAC ArA AAG cag cor rac ArG AGG GAG AAA rac AA TAT rTr CAC ccr ccr GCA CAC 149220. D__ Y__ IK__ GRCMR BK _ CK.__PH PPAH239CysHis motif 41500 TTG CAG GCC AAA ATC AlA GCT GCG CAG ccG CGG CcG CGG CCA CAG TCA TGA CTC aoT CGA 1559240IQAKI.KAA日P.RPRP日s#2551560CrG CCA AAG cA TGA AOC GAC CTC TCG Aag ca CTG TaG ACC roo CCT TTC ccc CTG GTG 16191620 CTC TTC ATC CTTr TAC CAA AGA GAC AAG CAC TTG AAA AAA GCA ATG GTA CCA aCG CaG TCT 16791680 TTA Acc ccA acG TCT rac ACT ACC AGC AGG CTC TCA CCA GCG CAC AGT TOC AGC AAC AC 17391740 CCcG cor TCA TTC CAA CAG GGT CAG TTT TGT GCA TGA CAC CCG CTA CCA GTA TTG TAC CCA 17991800 TGA rac ACA GCG CTA CGT CCG CCA CTG TCT CTG CAG CAA CAA CTC CTG CAA CAA OTG TCC 16591160 ccr TCG CAG CAA CAG CCA CAG CCA ATC AGA A TTC TQA AAT AAT CAG CAG A Cao MAT 19191920 GGA ATG cCA AGA ATC TaC ATT GAG AAT AAC TAA ACA TTG TTA CTG TAC ATA CTA rCC ToT 19791980 TTC CTC CTC AAT AGA ATT GCC ACA AAC TGC ATG CTA AAT AAA GAT GTA GTT CTT CTG GAC 20392040 AGA cCA CAA crC TAA GAA acr AGT acT GCT ATC TCA TAT ATG AGT ATT AAA TAT GGT ATG 20992100 cTr AGT ATA TTC CAA cct AAG ATA GTT AAc TAC CTG AGA CCA GCT GTG ATG TTT AAA GAC 21592160 ATA AAG QAT AAA GTT TAC TTT TAA AGG GTT TCT AAA CAT AGT TTC TGT CCT AGG AAT ATT 22192220 oTC TTA TCT CCA TAA CTA TAT AGC TGA TGC AGA AAG TCC AGC CAG TTt ACT cAT TrC QAT 22792280 TCA GMA TAT TTC AAA rTTr AGC AAT AAA CAA TTA GCA TTA oTT AAA AAA aAA ACA TAt TCC 29392340 AAG GOC AGG TTC GAT TCT AGC TCT AAT TAC mam pam rrr AT rar rra CTG GAT CAA A00 23992400 GTA TGT TTC ACT TCT TQA CAA TAT AAA TGC中国煤化工:CA nor AA ACC 24592460 GAT Acc TGT CCT GCA GGT CTA AA TTT GAAPAC rGG A CAC 25192520 ATC AAA GTG rGG TGT TTG GTT TOC CTG GAGMYHCNMH GiGA rI AOAST 287982580 CGA CCT GCA GGC ATG C2595Fig. 1. Nucleotides and deduced amino acid sequence of MBLL cDNA. The 4 CysHis motifs are underlined2Chinese Science Bulletin Vol. 45 No. 1January 2000(ii) Northern blot and RNA dot blot analysis. The Multiple Tissue Northern (MTNTM) Blotmembrane of mRNA from 8 different tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidneyand pancreas) was purchased from Clontech Ltd. 25 ng probe was randomly labeled with [ax 32P]dCTPby using Prime-a-GeneR Labeling System of Promega company. Hybridization was done according tothe Clontech' s manual.RNA dot blot hybridization was carried out by using Human RNA Master BlotTM (RNAs from 43adult tissues and 7 fetal tissues were dotted on the membrane) according to the Clontech's manual.2 Results( i ) Cloning of MBLL cDNA. Three integrated contigs of human ESTs were found throughcomparison of Drosophila mbl with human ESTs and Unigene database at NCBI by using TBLASTNprogram. The gene which is corresponding to one contig has been cloned and designated KIAA0428(GenBank Acc. AB007888). Primer pairs 15456, 15457 and 15454, 15455 were designed accordingto EST AA086138 and EST13608, respectively. Specific fragments were amplified by primers 15456,15457. 8 positive clones were obtained by cDNA library screening.The single clones were converted to pDR2 plasmid. The longest clone ME was submitted tosequencing. The nucleotide and deduced amino acid sequence are shown in fig.1. The gene wasdesignated MBLL (Muscleblind-like, GenBank Acc. AF061261). The length of the clone is 2595 bpwhich contains a 765-bp open reading frame encoding 255 amino acids. Molecular weight and pI of thededuced protein are 28.1 ku and 9.16 respectively. The ATG at 783- 785 was regarded as the startcodon. Several stop codons were found in upstream sequence of the start codon. The flanking sequenceof start codon coincides with Kozak's sequencel9] (-3, +4 were A and G respectively). There is notypical polyadenylation signal at its 3’end. Northern blot result showed that the 3’end sequence ofMBLL was not complete. But through the analysis of its ORF and homologous protein, the completeORF was obtained.(ii) Amino acid sequence analysis of MBLL. Homology comparison of MBLL amino acidsequence indicate that it is highly homologous to human KIAA0428, MBNL, Drosophila Mbl (splicedisoforms A, B and C) and C. elegans K02H8.1. At 269- 287, MBNL has 18 more amino acids thanKIAA0428. Their corresponding mRNA molecule should be different spliced isoforms of the samegene. Both Drosophila Mbl and C. elegans K02H8.1 contain 2 Cys;His zinc finger motifs. Humananalog MBLL and MBNL (including KIAA0428) contain 4 Cys;His zinc finger motifs. The first andthird ones are the same as those in Drosophila and C. elegans, which show typical CX7CX6CX3HCys,His structure, but the second and fourth ones are CX7CX4CX3H type which has 2 amino acidsabsence. Clustal W program was used to perform multiple alignment of these Cys;His zinc fingermotifs and result showed they are highly conserved in amino acid sequences (fig.2). The Cys,His zincmotifs in these proteins were similar to those in TIS11/NUP475/TTP protein family. The bindingactivity of mouse TIS 11/NUP475/TTP to zinc has been provedl!0By retrieving Prosite database, MBLL probably contains these motifs: 3 PKC phosphorylation15 1630 3145 4660 61681 hMBNL (1)LEVCREFQRG兀SRP D TECKFAHPSAS--C ON ENG RV /ACFDSLK GRCSREN- -CKYLHP PPHLKTOL 642 hMBLL(1) LEVCR OFQRG兀SRS DECKFAHP PAS--C ON ENGRV 1ACFDSLK GRC SREN--CKYLHP P HLKTOL 643 dMBLLEVCREFQRNNCSRO D ECKF AHPPAN--V EV QNGAVTAC rDS IK GRC MR DKPPCKYFHP P OHLK 0QL664 oK02H8. 1VEVCREF LRG OCARS DOECKFAHPPPN--V DN Q00RVTAC DSIK GRC TRENPKOKYLHP POH /KDOL 665 hMBNL(2) LEVCRE ORGMCMRG ENOCFAHPADS TMI OTNDNTVTV CMDrIK GRC SREK--CKYAHP PAHL 0AKI 666 hMBLL (2)LEVCREFQRGMCARG ETDC FAHPADS TMI OTSDNNVTV CMDYIK GRCMREK--CKYAHP PAHL 0AK166CyaH zincCCHfingersFig. 2. Multiple alignment of the CysH zinc motifs containin中国煤化工ters are conservative aminoacids. hMBNL (1) is the first and second Cys,H zinc motif of hy0HC N M H Gis the third and fourth Cys,Hzinc motif of human MBNL protein. hMBLL (1) is the first and second CysH zinc motit ot human MBLL protein. hMBLL (2)is the third and fourth Cys;H zinc motif of human MBLL protein. dMBL is two Cys,Hqs zinc motifs of Drosophila MBL protein.cK02H8.1 is the two Cys;H zinc motifs of C.elegans. K02H8.1 protein.Chinese Science BulletinVol.45No.1.January 20003sites (amino acid residues 56- -68, 170- 172, 176- 178), 4 CK2 phosphorylation sites (amino acidresidues 28一31, 30- -88, 77- -80, 206- 209), 2 tyrosine phosphorylation sites (amino acid residues 60-68, 225- 233) and 3 myrstication sites (amino acid residues 123- -128, 142- 147, 166- -171).Hydrophilic analysis by HOPP.THR program of PROSIS showed that there was a hydrophobicarea in the central area of MBLL. The N-terminal and C-terminal mainly consist of hydrophilic aminoacids (fig. 3).2-仆-1-050120180240300Amino acid residuesFig. 3. Hydrophilic analysis of MBLL amino acids.(li) Tissue expression analysis of MBLL. A 5.3 kb band was detected at 8 different adult tissuesby Northern blot and the amount is high. Hybridization by β-actin cDNA probe showed that the RNAloading amounts of these tissues are the same (fig. 4).RNA dot blot hybridization was performed withkb12.345678Human RNA Master BlotTM to detect the expressionin 43 adult and 7 fetal tissues. The result showed thatit was expressed in all these tissues, but the amount of二transcripts is different (fig. 5). .3 DiscussionCells in the Drosophila eye are determined byinductive signaling. Activation of the Ras/EGF-2.4 -receptor pathway causes induction of precursor cellsas photoreceptor neurons!"". This is followed by the1.35- -establishment of distinct photoreceptor subtypes,probably achieved by the expression of differentspecific factors' 124. mbl, (Musclebind) is autonomouslyrequired forphotoreceptor development. MutantB-actincells are recruited into developing ommatidea andinitiate neural differentiation， but they fail todifferentiate properly as photoreceptors')]. Since theFig. 4. Northem blot analysis of MBLL at 8 adult tssues.s translation product of MBLL is highly homologous to1, Heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletalDrosophila Mbl protein, MBLL may participate in themuscle; 7, kidney; 8, pancreas.development and functional maintenance of humaneye photoreceptors. The wide expression profile suggest that besides its function in eye development,MBLL should have other functions in different tissues. Through Unigene database retrieval, it wasfound that MBNL and KIAA0428 which are highly_ homoloons tn_ MRI I. also expressed in severaltissues. Drosophila mbl expresses in pharyngeal, visc中国煤化工ral nerve cored, and thelarval photoreceptor system..MHCNMHG4Chinese Science Bulletin Vol. 45 No. 1January 20001。234:678B中●。G●*●HFig. 5. The RNA dot blot analysis of MBLL at 50 tissues. (a) The tissue typesand positions of poly A+ RNAs and controls dotted on the positively chargednylon membranes; (b) the dot blot hybridization result of MBLL cDNA probe withthe membrane.Many proteins in different species contain Cys;His zinc motifs, but the biochemical function hasnot been identified. The mouse Nup475 protein, which contains 2 CysyHis zinc fingers, has beenproposed to be involved in transcriptional regulationl!o. The PIE-1 protein of C. elegans which containsthe same motif was regarded as the general inhibitor of somatic cell fatelsl. Some Cys;His zinc motifcontaining proteins have other functions; for example, U2AF35 is involved in pre-messenger RNAsplicing[141. An endoribonuclease activity has been demonstrated for the Drosophila Clipperl15]. The24_8caudatecerebralfrontalhippo-medullaAwhole brainamygdalacerebellumnucleuscortexlobecampusoblongataoccipitalsubstantiatemporalsub-thalamusspinalputamennegrathalamuscordskeletalCheartaortacolonbladderuterusprostatestomachmusclepituitaryadrenalthyroidsalivarymammaryDtestisovarypancreas .gland_glandkidneyliversmallspleenthymusperipherallymphboneintestineleukocytenodemarrowappendixlungtrachea _placentafetalfetal brainfetal heartfetal kidneyfetal liverfetal lungyeastE.coliE. colihumantotal RNAyeast tRNArRNADNAPoly r(A)Cgt-1DNA100 ng_ 100 ng500 ngmolecular mechanism of the bio-function of human MBLL and Drosophila Mbl has not been identified.They may be involved in the transcriptional regulation during some development procedure, such as thedevelopment of photoreceptors. They may bind to RNA molecule and regulate the metabolism of RNA.The two highly homologous genes, MBLL and MBNL (KIAA0428), may evolve from the samegene. Their highly conservative amino acid sequencInr fnnrions. These two genesmay fulfill their functional effect by different regulati中国煤化工Acknowledgments This work was supported by the State “86MHC N M H G19-02-02-01), the NationalNatural Science Foundation of China (Grant No.39830070) and the nauonar y75 ryect .ReferencesChinese Science BulletinVol. 45 No. 1January 200051. Miklos, G. L, Rubin, G M., The role of the genome project in determining gene function: insights from model organisms,Cell, 1996, 86: 521.2. Banfi, S., Borsani, G., Rossi, E. et al, Identification and mapping of human cDNAs homologous to Drosophila mutantgenes through EST database searching, Nature Genet., 1996, 13: 167.3. 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