ptpn12 msh6 ,and - hindawi publishing...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 902467 10 pageshttpdxdoiorg1011552013902467

Research ArticlemiR-1279 miR-548j miR-548m and miR-548d-5p Binding Sitesin CDSs of Paralogous and Orthologous PTPN12 MSH6 andZEB1 Genes

Anatoliy T Ivashchenko Assel S Issabekova and Olga A Berillo

National Nanotechnology Laboratory Al-Farabi Kazakh National University Almaty 050038 Kazakhstan

Correspondence should be addressed to Anatoliy T Ivashchenko a ivashchenkomailru

Received 27 February 2013 Revised 14 May 2013 Accepted 28 May 2013

Academic Editor Vassily Lyubetsky

Copyright copy 2013 Anatoliy T Ivashchenko et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Only PTPN12 MSH6 and ZEB1 have significant miR-1279 binding sites among paralogous genes of human tyrosine phosphatasefamilyDNAmismatch repair family and zinc finger family respectively AllmiRNAbinding sites are locatedwithinCDSs of studiedmRNAs Nucleotide sequences of hsa-miR-1279 binding sites with mRNAs of human PTPN12 MSH6 and ZEB1 genes encodeTKEQYE EGSSDE and GEKPYE oligopeptides respectively The conservation of miRNA binding sites encoding oligopeptideshas been revealed MRNAs of many paralogs of zinc finger gene family have from 1 to 12 binding sites coding the same GEKPYEhexapeptide MRNAs of PTPN12 MSH6 and ZEB1 orthologous genes from different animal species have binding sites for hsa-miR-1279 which consist of homologous oligonucleotides encoding similar human oligopeptides TKEQYE EGSSDE and GEKPYEMiR-548j miR-548m and miR-548d-5p have homologous binding sites in the mRNA of PTPN12 orthologous genes which encodePRTRSC TEATDI and STASAT oligopeptides respectively All regions of miRNA are important for binding with the mRNA

1 Introduction

Posttranscriptionally miRNAs regulate the expression ofgenes involved in organism development [1] metabolism [2]cell cycle apoptosis [3] carcinogenesis [4] protein-proteininteractions [5] and so forth The specific functions of mostidentified miRNAs remain unknown although some targetshave been established Computational algorithms have beendeveloped to predictmiRNA targets [6] For animalmiRNAstarget prediction is an issue because miRNAs can bind totarget genes without perfect complementarity [7] Only somepredicted miRNA binding sites had been experimentallyverified [8]

Many pieces of software initially used complementarityto identify potential binding sites Subsequent filtering stepswere based on thermodynamics binding site structuresand site conservation across species [9] The false-positiverate of predicting miRNA targets has resulted in the devel-opment of several algorithms miRanda (httpwwwmicro-rnaorgmicrornahomedo) [10] TargetScan (httpwwwtargetscanorg) [11] and PicTar (httppictarmdc-berlinde) [12] Other algorithms apply thermodynamics as

the initial requirement for selection of interaction sitesbetween miRNAs and their targets DIANA-microT(httpdianacslabecentuagr) [13] and RNAhybrid (httpbibiservtechfakuni-bielefelddernahybrid) [14] belongto this type of software We have used a local RNAHybridsoftware to identifymiRNA target sites and have attempted toexplain the benefits of this software [15] The software allowsthe determination of the most energetically favourableinteractions of small RNAs to mRNAs in all regions51015840untranslated regions (51015840UTR) coding domain sequences(CDS) and 31015840untranslated regions (31015840UTR) The majority ofon-line pieces of software predict miRNA target sites only inthe 31015840UTR and most of the characterised miRNA bindingsites occur in this region [16] Experimental investigationsrevealed the occurrence of 51015840UTR and CDSmiRNAs bindingsites [16ndash18] According to another study human mRNAscontain many strong sites with 13 or more base pairingswithout bulges in the middle [19]

Previously we had revealed miRNA binding sites in 54mRNAs of oncogenes including PTPN12 MSH6 and ZEB1that had sites in CDSs with a high Δ119866Δ119866

119898ratio [19] These

genes are involved in the development of breast cancer [20]

2 BioMed Research International

colorectal cancer [21] lymphoma [22] and esophageal cancer[23]The PTPN12 gene encodes a protein from tyrosine phos-phatase family It participates in different cellular processesincluding cell growth the mitotic cycle and oncogenic trans-formation [24]MSH6 is a DNAmismatch repair protein thatparticipates in the recognition of mismatched nucleotidesprior to their repair [25] Mutations in this gene have beenassociated with hereditary nonpolyposis colon cancer andendometrial cancer [26] The ZEB1 gene encodes a proteinwhich belongs to a large family of zinc finger transcriptionalfactors [27] Identification of the role of miRNA in theregulation of the gene and the gene set expression is animportant problem Thus we have examined the presence ofmiR-1279 target sites in paralogous genes for PTPN12 MSH6and ZEB1

MiRNA sites are localized within the 51015840UTR CDS and31015840UTR of mRNA [28] Indeed about half of all miRNAbinding sites are located in the protein-coding region [29]suggesting that studies should pay much attention to thesesites The nucleotide placement of these binding sites codingthe amino acid sequencemay raise important questions aboutbinding sites localized in the CDSs

To reduce the number of false positives target sitesmay be validated by conservation of sites across speciesTo verify predicted sites by RNAHybrid we have examinedconservation of these sites across orthologous genes ofdifferent organisms Significant sites in human genes havebeen investigated in PTPN12 MSH6 and ZEB1 orthologuesInvestigating these problems is necessary to establish the roleof miRNA in the regulation of single gene and gene setsexpressions

In present work we analysed the conservation of miR-1279 miR-548m miR-548j and miR-548d-5p binding sitesinorthologues and paralogues of the PTPN12 MSH6 andZEB1 genes to verify these interactions

2 Materials and Methods

Investigation objects were mRNAs of PTPN12 MSH6and ZEB1 human genes their orthologs and paralogs(Supplementary Tables S1 S2 in Supplementary Materialavailable online at httpdxdoiorg1011552013902467) inAnolis carolinensis (Aca) Ailuropoda melanoleuca (Ame)Bos taurus (Bta) Cricetulus griseus (Cgr) Cavia porcel-lus (Cpo) Callithrix jacchus (Cja) Canis lupus familiaris(Clf) Danio rerio (Dre) Equus caballus (Eca) Gallus gal-lus (Gga) Homo sapiens (Hsa) Loxodonta africana (Laf)Macaca mulatta (Mml) Monodelphis domestica (Mdo)Meleagris gallopavo (Mga) Mus musculus (Mmu) Nomascusleucogenys (Nle) Ornithorhynchus anatinus (Oan) Oryctola-gus cuniculus (Ocu) Oreochromis niloticus (Oni) Otolemurgarnettii (Oga) Pan paniscus (Ppa) Papio anubis (Pan)Pan troglodytes (Ptr) Pongo abelii (Pab) Rattus norvegi-cus (Rno) Saimiri boliviensis (Sbo) Sarcophilus harrisii(Sha) Sus scrofa (Ssc) Taeniopygia guttata (Tgu) Xeno-pus laevis (Xla) and Xenopus tropicalis (Xtr) Nucleotideand amino acid sequences were accessed from GenBank

(httpwwwncbinlmnihgov) Nucleotide sequence of hsa-miR-1279 hsa-miR-548m and hsa-miR-548j were receivedfrom miRBase (httpwwwmirbaseorg)

Free energy (Δ119866) position of potential binding sites andinteraction schemes were calculated by RNAhybrid 21 soft-ware (httpbibiservtechfakuni-bielefelddernahybrid)The E-RNAhybrid software (httpsitesgooglecomsitemalaheeneesoftware) was used to compute the ratio ofΔ119866Δ119866

119898and 119875value Δ119866Δ119866

119898value equalled 75 and

more was used as comparative criterion of miRNA andmRNA interaction force Δ119866Δ119866

119898value shows degree of

complementarity of each miRNA site in target gene Δ119866value and its standard deviation were used to determinesignificance level of miRNA binding sites with mRNAInteraction energies (Δ119866

119898) of hsa-miR-1279 hsa-miR-

548m hsa-miR-548 j and hsa-miR-548 d-5 p with theirperfectly complementary sequence were calculated andequaled minus118 kJmoL minus139 kJmoL minus161 kJmoL andminus148 kJmoL consequently At first miRNA binding siteswere found for human mRNAs and then they were revealedin homologous and paralogous genes of studied animalsDiagrams of nucleotide and protein variabilitywere producedby WebLogo software (httpweblogothreeplusonecom)

3 Results

31 Features of hsa-miR-1279 Binding Sites in mRNAs ofParalogous and Orthologous PTPN12 Genes To investigatethe role ofmiR-1279 in the regulation of tyrosine phosphatasefamily genes we attempted to elucidate target binding sitesamong them We identified the PTPN12 gene as a reliabletarget of hsa-miR-1279 with binding energy of minus1021 kJmoLand a Δ119866Δ119866

119898value of 865 hsa-miR-1279 consists of

17 nucleotides 15 of these nucleotides are perfectly comple-mentary to the binding site of human PTPN12 mRNA WeconsideredmiRNAbinding siteswithΔ119866Δ119866

119898values greater

than 75 All sites with lower values were determined to beweak sites and were not consideredThe hybridization energyof miR-1279 with the mRNA of tyrosine phosphatase familygenes except that of PTPN12 varied from minus677 kJmoL tominus895 kJmoL Corresponding Δ119866Δ119866

119898values ranged from

574 to 750 Thus only PTPN12 was considered Thenucleotide sequence of the binding site in PTPN12 encodedthe TKEQYE hexapeptide which was not a conserved aminoacid sequence in any other tyrosine phosphatase familyConsequentlymiR-1279may efficiently regulate onlyPTPN12gene expression

Furthermore we investigated the conservation of themiR-1279 binding site in orthologous genes The humanPTPN12 gene has 23 orthologues in different animals Fordetermination of the conservation of the relevant site weverified the nucleotide sequence of the binding site Thenucleotide sequences of these sites in the mRNA of theinvestigated genes are presented in Table 1 Sequences of themRNAPTPN12 binding sites in all animalswere homologousand nucleotides in first and second positions of the codonswere conserved (Figure 1(a)) ACGAAGGAGCAGUAUGAAoligonucleotide of the interaction site in the CDS encoded the

BioMed Research International 3

Table 1 Characteristics of miR-1279 binding sites in CDSs of PTPN12 orthologous genes

Hsa 788 865Ptr 788 865Cgr 836 879Ppa 836 865Aca 836 745Ame 840 833Bta 840 833Cja 836 631Clf 836 830Dre 828 716Eca 447 830Gga 788 865Laf 788 865Mdo 908 631Mga 836 865Mmu 788 865Nle 677 865Oan 479 879Ocu 479 865Oga 944 865Oni 765 830Pab 677 865Pan 836 865Sbo 836 865Rno 479 879Tgu 815 865Xla 836 713Xtr 788 865

Object Region of CDS inPTPN12orthologous genes

Position inCDS nt

Δ119866Δ119866m

Note the dots are identical nucleotides or amino acids in data presented in Tables 1ndash6

TKEQYE hexapeptide which was identical in all orthologousproteins of the studied species (Table 1) Replacements inthe third position of the codon in described oligonucleotidesequences did not change the amino acid content Thisoligopeptide was located from positions13 to 18 in theconserved amino acid sequences of tyrosine phosphataseorthologues (Figure 1(b)) These data indicate that the mainfunction of this site is gene regulation by miR-1279 All miR-1279 interaction sites in PTPN12 orthologues correspondedto an open frame Replacements of one or several nucleotidesin the third position of the codon led to decreased bindingenergies but did not change the location of the miR-1279binding site Interactions between hsa-miR-1279 and themRNA of PTPN12 orthologues showed significance levelsof Δ119866Δ119866

119898values ranging from 713 to 879 (Table 1)

MiRNA binding sites were identical in 11 species frommammals to amphibians

The binding energy of orthologous mRNA with orthol-ogous miRNA may vary if the sequences of orthologousmiRNAs in animals differ from that of hsa-miR-1279 Itis possible that the interactions of hsa-miR-1279 with the

mRNA of A carolinensis C jacchus D rerio M domesticaand X laevis tyrosine phosphatase orthologues are lesseffectiveThese data confirm that this binding site exists fromthe early stages of evolution and did not change for tens tohundreds of millions of years

32 Features of hsa-miR-1279 Binding Sites in mRNAs ofParalogous and Orthologs MSH6 Genes The human genomeencodes several DNA mismatch repair proteins MSH2MSH3MSH4MSH5MSH6MLH1 andMLH3 We did notfind conserved strong miR-1279 binding sites in the mRNAsof these genes with the exception of theMSH6 gene amongall consideredDNAmismatch repair proteins NonconservedmiR-1279 interaction sites in these genes hadΔ119866Δ119866

119898values

ranging from 620 to 805 and only the MLH3 genehad a significant binding site (Δ119866Δ119866

119898value is 73) These

data confirmed the specific binding of miR-1279 withMSH6mRNA among genes of the DNA mismatch repair family

The hsa-miR-1279 binding site in human MSH6 mRNAhad perfect complementary nucleotides with a singlenucleotide bulge including 4 G-U base pairs in spite of


210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 39984005998400

(a)

20(s

ites)

N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C

(b)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 39984005998400

(c)

2

0(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(d)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(e)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(f)

2

1

0

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 39984005998400

(g)

210(

sites

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(h)

2

0

3

1(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(i)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(j)

3210

(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12

(k)

210

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(l)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

(m)

Figure 1 Fragments of nucleotide and amino acid sequences where miRNA binding sites are located The variability of nucleotides in miR-1279 binding sites in mRNA of PTPN12 (a) MSH6 (c) and ZEB1 (e) orthologous genes and the variability of amino acids of PTPN12 (b)MSH6 (d) and ZEB1 (f) orthologous proteins miR-1279 binding site encode TKEQYE (b) EGSSDE (d) and GEKPYE (f) oligopeptides(g) the variability of nucleotides in miR-1279 binding sites of zinc-finger transcriptional factors family (h) (j) and (l)mdashthe variability ofnucleotides in 548j 548m and miR-548d-5p binding sites in mRNA of PTPN12 respectively (i) (k) and (m) the variability of amino acidsof PTPN12 orthologous proteins in regions with PRTRSC EATDI and STASAT oligopeptides respectively

its high Δ119866Δ119866119898

value (894) and binding energy(minus1054 kJmoL) The GAAGGAAGCAGUGAUGA oligonu-cleotide sequence in the binding site of MSH6 mRNA(Figure 1(c)) encoded the EGSSDE hexapeptide inMSH6 protein (Table 2) We observed 34 orthologuesfor human MSH6 and miR-1279 binding sites with highcomplementarily levels in 15 orthologues Oligonucleotidesof these sites and corresponding oligopeptides are shownin Table 2 MSH6 protein from 13 mammals and humancontained completely homologous EGSSDE hexapeptidesand the corresponding nucleotide sequences in mRNAbinding sites exhibited high homology There was nucleotidevariability in the third position of the glutamic acid codons

at the beginning of the EGSSDE hexapeptide situated frompositions 13 to 30 in the highly conserved protein regionof MSH6 (Figure 1(d)) These data suggest the functionalimportance of the miR-1279 binding site in the regulation ofMSH6 gene expression

All binding sites in MSH6 orthologous mRNA corre-sponded to an open reading frame Replacement of oneor several nucleotides in the third position of codons ledto diminished hybridization energy but did not change thelocation of the hsa-miR-1279 binding site Such replacementsdid not influence the amino acid composition properties ofthe protein The Δ119866Δ119866

119898value of interaction sites varied

from 876 to 894 (Table 2)


33 Features of hsa-miR-1279 Binding Sites in mRNAs ofHuman ZNF Family and ZEB1 Orthologous Genes The ZEB1gene is a member of the large ZNF gene family of transcrip-tion factors We searched for miR-1279 binding sites in theCDSs of several genes from this family ZNF552 and ZNF790mRNAs had miR-1279 sites where the Δ119866Δ119866

119898values

equalled 894 and 879 respectively Nucleotide sequencesof the binding sites in these 2 genes shared high homologyand encoded a GEKPYE oligopeptide In fact while manygenes of this family had the GEKPYE oligopeptide not allwere targets for miR-1279 There were no significant bindingsites for hsa-miR-1279 in ZNF91 ZNF148 ZNF208 ZNF232ZNF236 ZNF423 ZNF495B ZNF509 ZNF521 ZNF729ZNF776 ZNF768 ZNF853 ZNF857A and ZNF865 geneswithin the CDS and these genes did not encode the GEKPYEoligopeptide

Proteins encoded by ZEB2 ZNF8 ZNF70 ZNF84ZNF140 ZNF454 ZNF461 ZNF475 ZNF529 ZNF576ZNF594 ZNF713 ZNF751 and ZNF755 genes had thisoligopeptide in structure but the corresponding oligonu-cleotideswere boundbyhsa-miR-1279with lowhybridizationenergies These oligonucleotides differed from the humanZEB1 gene sequence by replacements in the third positions ofseveral codons (Figure 1(g)) These genes have between 1 and7 sites encoding the GEKPYE hexapeptide We assume thatif miR-1279 would be bound to these sequences gene expres-sion of these proteins would be inhibited The expression ofmajority of the genes in the zinc-finger transcription factorfamily was not regulated by miR-1279

The strongest binding site in genes of the zinc-fingertranscription factor family was found in ZEB1 mRNA Thebinding energy of miR-1279 with ZEB1 mRNA was 90 ofthe Δ119866

119898 and 16 out of 17 nucleotides from the hsa-miR-1279

sequence exhibited total complementarity Thus it is sug-gested that they may interact in vivo To verify the miR-1279binding site in theZEB1 gene we investigated the interactionsbetween hsa-miR-1279 and mRNAs of ZEB1 orthologuesfrom 12 animal species The human ZEB1 gene has 16 iden-tified orthologues but only 12 of them retained this hsa-miR-1279 binding site and had conserved amino acid sequencesin the binding region The nucleotide sequence of the miR-1279 binding site (GGAGAGAAGCCAUAUGA) had highhomology between these 12 orthologues (Figure 1(e)) Thethird nucleotide of these codons sequences encoded changesin tyrosine and glutamic acid but these changes did notinfluence the encoding of the GEKPYE oligopeptide Thisoligopeptidewas localized in the conserved region of ortholo-gous proteins (Figure 1(f)) Characteristics of the interactionsbetween hsa-miR-1279 and ZEB1 orthologue mRNAs ofdifferent animals are shown in Table 3 This binding site waswell conserved among mammals and birds but was found inamphibians with less accuracy

34 Features of hsa-miR-548j Binding Sites in mRNAs ofPTPN12 Orthologous Genes PTPN12mRNA contained nearperfect binding sites for several miRNAs (miR-1279 miR-548j and miR-548m) In the first part of this study wevalidated miR-1279 binding site conservation across species

Furthermore we considered the conservation of miR-548jinteraction sites in PTPN12 mRNA MiR-548j has intronicorigins (intron 9 in theTPST2 gene)Thenucleotide sequenceof the miR-548j binding site in the CDS of PTPN12 mRNAexhibited low variability and was present in all investigatedorganisms from mammals to fish (Figure 1(h)) Character-istics of these interactions are presented in Table 4 TheΔ119866119898value of miR-548j with a completely complementary

sequence equalled minus161 kJmoL The nucleotide sequence ofthe miR-548j binding site corresponded to the PRTRSColigopeptide This hexapeptide did not change across 16species of mammals reptiles and birds but exhibited somechanges in 4 species of fishes and amphibians (Figure 1(i))Such conservation of revealed binding sites indicates thepossibility of regulator role of miR-548j in PTPN12 geneexpression

The third miRNA that binds to PTPN12mRNA was miR-548mThis binding site showed a lower level of conservationthan miR-1279 and miR-548j binding sites MiR-548m inter-acted with the site encoding a conserved TEATDI hexapep-tide in 7 species (Figure 1 (k)) A homologous oligopeptidefrom other animal species exhibited mismatches in 1 or2 amino acids but had a similar position in a conservedregion of the protein The Δ119866

119898value of miR-548m with a

perfectly complementary sequence equalled minus1394 kJmoLCharacteristics of miR-548m binding sites are presented inTable 5 and showed a high level of conservation In fact thebinding site only had perfect conservation across 4 inves-tigated species (chimpanzee gibbon horse and elephant)The Δ119866Δ119866

119898values vary from minus751 to minus808 (Table 5)

Consequently the effect of miR-548m on PTPN12 mRNAexpression was less than those of miR-1279 and miR-548j

35 Features of hsa-miR-548d-5p Binding Sites in mRNAs ofPTPN12OrthologousGenes MiR-548d-5p had another bind-ing site in PTPN12mRNAThe binding energy of this site waslower than in the miR-548j and miR-548m binding sites butthe nucleotide sequence in this site exhibited conservation inorthologous genes (Table 6) The amino acid sequences up-and downstreamof the hexapeptide STASATof PTPN12werevariable (Figure 1 (m))The nucleotide sequences of the miR-548d-5p binding sites and PTPN12mRNA were homologous(Figure 1(l)) however nucleotides in the third codon positionwere variable and thus these variable nucleotides did notcause changes in the amino acids coding of hexapeptideSTASAT in some cases

36 Nucleotide Interactions of hsa-miR-1279 Binding Sites withmRNAs of PTPN12 MSH6 and ZEB1 Next we investigatedthe regions of the miRNA that are important for target generegulation Binding sites may be different according to theprimary contribution of the specific region of miRNA tothe hybridization energy We observed 3 types of miRNAcontributions (1) the contribution of 51015840-end dominated (2)the contribution of the central region dominated and (3)the contribution of 31015840-end dominated (Table 7) A schematicrepresentation of the nucleotide sequences of hsa-miR-1279


Table 2 Characteristics of miR-1279 binding sites in CDSs of MSH6 orthologous genes and the variability of amino acids of MSH6orthologous proteins in regions with EGSSDE oligopeptide

HsaMmlPanNlePabPpaPtrSboCpoClfEcaMdoMmuOcuShaSsc

812 894812 894812 894602 894818 894629 894929 894809 894806 894578 894758 894

1019 876812 883809 894998 876815 894

Object Region of CDS inMSH6orthologous genes

Region of MSH6orthologous proteins

Position inCDS nt

Δ119866Δ119866m

Table 3 Characteristics of miR-1279 binding sites in CDSs of ZEB1 orthologous genes

Hsa 741 894Nle 741 894Ppa 741 894Ptr 741 894Sbo 741 894Ame 810 894Bta 792 894Cja 792 894Pab 792 894Clf 588 894Mml 750 894Oga 729 894Pan 770 894Gga 789 894Mmu 730 741Sha 741 894Xla 798 741Xtr 732 741

Object Region of CDS inZEB1orthologous genes

Position inCDS nt

Δ119866Δ119866m

binding sites in the mRNA of PTPN12 MSH6 and ZEB1from different animals is shown in Table 7 The miR-1279binding site in the PTPN12 gene of 14 animal species was51015840-dominant and evolutionary conserved PTPN12 mRNAsfrom A melanoleuca C lupus E caballus and O niloticusalso had perfect complementarity in 51015840-end of miR-1279 buthad a reduced number of conserved nucleotides than thefirst gene group and had several mismatches in the targetregion mRNAs of MSH6 from 11 animal species had 31015840-dominant miR-1279 sites and were highly homologous in all

species The 31015840-end of miR-1279 was the primary contributorin these binding sites MiR-548j was bound to the mRNAof the investigated species through the central region or the31015840-end in O niloticus and D rerio (Table 7) As shown inTable 7 miR-548m and miR-548d-5p interacted predomi-nantly with the 31015840-end and in several cases the central region(A carolinensis C jacchus Gallus gallus M gallopavo Oniloticus and P abelii) All described data confirmed that thenucleotide sequences ofmRNA sitemay bindwith all miRNAregions


Table 4 Characteristics of miR-548j binding sites in CDSs of PTPN12 orthologous genes and the variability of amino acids of PTPN12orthologous proteins in regions with PRTRSC oligopeptide

HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu


Region of PTPN12orthologous proteins

6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m

Table 5 Characteristics of miR-548m binding sites in CDSs of PTPN12 orthologous genes and the variability of amino acids of PTPN12orthologous proteins in regions with EATDI oligopeptide

HsaPanPpaPtrEcaNleClfLafCgrOcu

Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m

Table 6 Characteristics of miR-548d-5p binding sites in CDSs of PTPN12 orthologous genes and the variability of amino acids of PTPN12orthologous proteins in regions with STASAT oligopeptide

Hsa 1878 659Pan 1878 659Cja 1875 659Nle 1872 659Ptr 1872 659

Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645

Ame 1881 659Bta 1011 597Clf 1494 659Eca 1806 552Gga 1872 555Laf 1884 651

Mdo 1950 594Mga 1872 606

Mmu 1872 594Ocu 1860 659Oga 1923 659Rno 1860 499Sha 1920 594Tgu 2146 569Xla 1803 501Xtr 1821 501



Position inCDS nt

Δ119866Δ119866m


Table 7 Schemes of hsa-miR-1279 biding with mRNA of PTPN12 MSH6 and ZEB1 orthologous genes and schemes of hsa-miR-548j hsa-miR-548m and hsa-miR-548d-5p biding with PTPN12 orthologous gene

miR-1279

Cgr Gga Hsa Laf MgaMmu Nle Oan Ocu OgaPab Pan Ppa Ptr RnoSbo Tgu Xla Xtr

miR-1279

Clf Cpo Eca Hsa MdoMml Mmu Nle Ocu PabPan Ppa Ptr Sbo Sha Ssc

miR-1279

Ame Cja Clf Bta GgaHsa Mml Nle Oga PabPan Ppa Ptr Sha Sbo

miR-548j

Ame Bta Cja Clf EcaHsa Laf Pab Pan PpaPtr Sbo

miR-548m

Ame Cgr Clf Eca HsaLaf Nle Ocu Pan Ppa Ptr

miR-548d-5p

Cja Hsa Nle Ocu OgaPab Pan Ppa Ptr SboAme Clf

mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

Scheme of binding site Objects with samebinding site

Note N any nucleotidemdashA G U or C nucleotides

4 Discussion

Previously it was shown that 54 human mRNAs involved inoncogenesis contained strong miRNA binding sites withintheir CDSs that were predicted by RNAhybrid [19] Inter-actions between mRNAs and miRNAs in binding siteswere selected using Δ119866Δ119866

119898values allowing us to identify

miRNA interactions with near perfect complementarity Weexamined the variation in sequences of binding sites and havebeen able to show good conservation of strong binding sitesamong the organisms we have investigated

It was predicted that miR-127 was 9 bound to mRNAof PTPN12 MHS6 and ZEB1 in sites encoding 3 differentoligopeptides (Tables 2 4 and 6) miRNA binding sitesmay correspond to 3 open reading frames Interactionsbetween PTPN12 MSH6 and ZEB1 mRNAs and miR-1279as well as interactions between PTPN12 mRNA and miR-548j miR-548m and miR-548d-5p correspond to differentopen reading frames in their mRNA targets The ability ofmiRNA binding sites to encode 1 oligopeptide suggests thestable influence of miRNA on mRNA during evolution

Similar conservation of nucleotide regions in CDSs ofstudied mRNAs and corresponding amino acid of proteinshas been established for human APC BAD EPHB2 andMMP2 genes (unpublished data) MiRNA binding sites in

plant proteins encode regions of mRNAs in paralogous genesof the SPL family with high homology of oligonucleotidesand corresponding hexapeptides (unpublished data) Thenucleotide sequences adjacent to binding sites and to thecorresponding amino acids have high variability (Figure 1)Highly homologous miRNA binding sites of orthologousgenes and conservative oligopeptides of corresponding pro-teins have been established

The binding sites of several miRNAs are located in the31015840UTRs of some orthologous genes in theDrosophila genomeand have highly homologous nucleotide sequences [30] Allnucleotides of miRNAs that participate in forming bindingsites with mRNA in plants are highly conserved and notonly in seeds [31] These data prove the high conservatismof many miRNAs and show the importance of all nucleotidesequences ofmiRNAswith respect to binding tomRNAsThepresence of many target genes for a single miRNA suggeststhe functional connection of these genes According to thedata collected for the zinc-finger transcription factor familythe presence of oligopeptides that associate with the miRNAbinding site in mRNA is not necessarily sufficient for miRNAsite prediction Changes in the third position of codons maycause weakening of the miRNA binding site or result in lackof binding ability We have found this effect in many genesfrom the zinc-finger familyMutations in the third position of


codons can lead to loss of functional importance of miRNAbinding

Target site conservation is one of the primary predic-tion validation methods We have used this prediction toevaluate several factors Investigation of 3 different genefamilies demonstrated that miR-1279 efficiently regulatedonly 1 gene from the tyrosine phosphatase family and DNAmismatch repair family Furthermore miR-1279 affectedseveral mRNAs from paralogous genes of the zinc-fingertranscription factor family

Single miRNAs can also affect gene sets by conservationof the target site in orthologous genes MiR-1279 has beenshown to have a high probability of regulating PTPN12MHS6 and ZEB1 Estimation of the degree of binding ofmiRNA with orthologous mRNAs has demonstrated that thedegree of complementarity increases from fish and amphib-ians to mammals and is identical across primates Similartrends have been identified in the ZEB1 gene Additionallyseveral miRNAs have been confirmed to affect a single geneThese data support that miR-1279 miR-548j miR-548mand miR-548d-5p may potentially regulate PTPN12 geneexpression

5 Conclusion

High conservation of binding sites in orthologous geneshas proven the importance and antiquity of miRNA inter-actions with mRNAs of target genes The expression ofsome paralogous genes can be regulated by a single miRNAHowever repression of the expression of entire gene familiesvia 1 miRNA is unlikely The expression of one gene canbe regulated by several miRNAs presupposing multiplemethods of gene regulation that include dependence on theexpression of intronic miRNAs Establishment of multiplemiRNA interactions with mRNAs is a complex problembut it is necessary for definite regulation of gene expressionthroughmiRNAsThe power ofmiRNA linkage withmRNAsin CDSs can change at the expense of nucleotide replacementin the third position of the codons This phenomenon hasbeen clearly shown in miR-1279 binding sites in the mRNAsof human ZNF family genes

Acknowledgments

This study was supported by grant from the Ministry of Edu-cation and Science Kazakhstan Republic The authors wouldlike to thank V Khailenko for the software E-RNAhybrid 21

References

[1] R C Lee R L Feinbaum and V Ambros ldquoThe C elegansheterochronic gene lin-4 encodes small RNAs with antisensecomplementarity to lin-4rdquo Cell vol 75 no 5 pp 843ndash854 1993

[2] P Xu S Y Vernooy M Guo and B A Hay ldquoThe DrosophilamicroRNA mir-14 suppresses cell death and is required fornormal fat metabolismrdquo Current Biology vol 13 no 9 pp 790ndash795 2003

[3] J Brennecke D R Hipfner A Stark R B Russell andS M Cohen ldquobantam encodes a developmentally regulated

microRNA that controls cell proliferation and regulates theproapoptotic gene hid in Drosophilardquo Cell vol 113 no 1 pp25ndash36 2003

[4] M Liu and H Chen ldquoThe role of microRNAs in colorectalcancerrdquo Journal of Genetics and Genomics vol 37 no 6 pp 347ndash358 2010

[5] J Krol I Loedige and W Filipowicz ldquoThe widespread reg-ulation of microRNA biogenesis function and decayrdquo NatureReviews Genetics vol 11 no 9 pp 597ndash610 2010

[6] T M Witkos E Koscianska and W J Krzyzosiak ldquoPracticalaspects of microRNA target predictionrdquo Current MolecularMedicine vol 11 no 2 pp 93ndash109 2011

[7] N Rajewsky ldquomicroRNA target predictions in animalsrdquoNatureGenetics vol 38 supplement 1 pp S8ndashS13 2006

[8] C Zhao C Huang T Weng X Xiao H Ma and L LiuldquoComputational prediction of MicroRNAs targeting GABAreceptors and experimental verification of miR-181 miR-216andmiR-203 targets inGABA-A receptorrdquoBMCResearchNotesvol 5 no 91 pp 1ndash8 2012

[9] P Lekprasert MMayhew andU Ohler ldquoAssessing the utility ofthermodynamic features formicroRNA target prediction underrelaxed seed and no conservation requirementsrdquo PLoS ONEvol 6 no 6 Article ID e20622 2011

[10] A J Enright B John U Gaul T Tuschl C Sander and D SMarks ldquoMicroRNA targets inDrosophilardquoGenomeBiology vol5 no 1 p R1 2003

[11] B P Lewis C B Burge and D P Bartel ldquoConserved seedpairing often flanked by adenosines indicates that thousandsof human genes are microRNA targetsrdquo Cell vol 120 no 1 pp15ndash20 2005

[12] D Grun Y Wang D Langenberger K C Gunsalus andN Rajewsky ldquoMicroRNA target predictions across sevendrosophilo species and comparison to mammalian targetsrdquoPLoS Computational Biology vol 1 no 1 Article ID e13 2005

[13] M Kiriakidou P T Nelson A Kouranov et al ldquoA com-bined computational-experimental approach predicts humanmicroRNA targetsrdquo Genes and Development vol 18 no 10 pp1165ndash1178 2004

[14] A Stark J Brennecke R B Russell and S M Cohen ldquoIdenti-fication of Drosophila microRNA targetsrdquo PLoS Biology vol 1no 3 Article ID E60 2003

[15] M Rehmsmeier P Steffen M Hochsmann and R GiegerichldquoFast and effective prediction of microRNAtarget duplexesrdquoRNA vol 10 no 10 pp 1507ndash1517 2004

[16] M Schnall-Levin O S Rissland W K Johnston N PerrimonD P Bartel and B Berger ldquoUnusually effective microRNAtargeting within repeat-rich coding regions of mammalianmRNAsrdquo Genome Research vol 21 no 9 pp 1395ndash1403 2011

[17] L DHurst ldquoPreliminary assessment of the impact ofmicrorna-mediated regulation on coding sequence evolution in mam-malsrdquo Journal of Molecular Evolution vol 63 no 2 pp 174ndash1822006

[18] X Zhou X Duan J Qian and F Li ldquoAbundant conservedmicroRNA target sites in the 51015840-untranslated region and codingsequencerdquo Genetica vol 137 no 2 pp 159ndash164 2009

[19] A S Issabekova O A Berillo M Regnier and A TIvashchenko ldquoInteractions of intergenic microRNAs withmRNAs of genes involved in carcinogenesisrdquo Bioinformationvol 8 no 11 pp 513ndash518 2012

[20] S Volinia M Galasso M E Sana et al ldquoBreast cancer signa-tures for invasiveness andprognosis defined bydeep sequencing


of microRNArdquo Proceedings of the National Academy of Sciencesof the United States of America vol 109 no 8 pp 3024ndash30292012

[21] G Kaur A Masoud N Raihan M Radzi W Khamizar andL S Kam ldquoMismatch repair genes expression defects andassociation with clinicopathological characteristics in colorec-tal carcinomardquo Indian Journal of Medical Research vol 134 no2 pp 186ndash192 2011

[22] T Ripperger C Beger N Rahner et al ldquoConstitutional mis-match repair deficiency and childhood leukemialymphoma-report on a novel biallelic MSH6 mutationrdquo Haematologicavol 95 no 5 pp 841ndash844 2010

[23] A Feber L Xi J D Luketich et al ldquoMicroRNA expressionprofiles of esophageal cancerrdquo Journal of Thoracic and Cardio-vascular Surgery vol 135 no 2 pp 255ndash260 2008

[24] T Sun N Aceto K L Meerbrey et al ldquoActivation of multipleproto-oncogenic tyrosine kinases in breast cancer via loss of thePTPN12 phosphataserdquo Cell vol 144 no 5 pp 703ndash718 2011

[25] A KWin J P Young NM Lindor et al ldquoColorectal and othercancer risks for carriers and noncarriers from families witha DNA mismatch repair gene mutation a prospective cohortstudyrdquo Journal of Clinical Oncology vol 30 no 9 pp 958ndash9642012

[26] MOllikainenWM Abdel-Rahman AMoisio et al ldquoMolecu-lar analysis of familial endometrial carcinoma a manifestationof hereditary nonpolyposis colorectal cancer or a separatesyndromerdquo Journal of Clinical Oncology vol 23 no 21 pp4609ndash4616 2005

[27] Y Arima H Hayashi M Sasaki et al ldquoInduction of ZEBproteins by inactivation of RB protein is key determinant ofmesenchymal phenotype of breast cancerrdquo Journal of BiologicalChemistry vol 287 no 11 pp 7896ndash7906 2012

[28] I Lee S S Ajay I Y Jong et al ldquoNew class of microRNA targetscontaining simultaneous 51015840-UTR and 31015840-UTR interaction sitesrdquoGenome Research vol 19 no 7 pp 1175ndash1183 2009

[29] A S Issabekova O A Berillo V A Khailenko S A Atam-bayeva M Regnier and A T Ivachshenko ldquoCharacteristicsof intronic and intergenic human miRNAs and features oftheir interaction with mRNArdquo World Academy of ScienceEngineering and Technology no 59 pp 63ndash66 2011

[30] S Miura M Nozawa andM Nei ldquoEvolutionary changes of thetarget sites of two MicroRNAs encoded in the Hox gene clusterof Drosophila and other insect speciesrdquo Genome Biology andEvolution vol 3 no 1 pp 129ndash139 2011

[31] M Nozawa S Miura and M Nei ldquoOrigins and evolution ofmicroRNA genes in Drosophila speciesrdquo Genome Biology andEvolution vol 2 no 1 pp 180ndash189 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


colorectal cancer [21] lymphoma [22] and esophageal cancer[23]The PTPN12 gene encodes a protein from tyrosine phos-phatase family It participates in different cellular processesincluding cell growth the mitotic cycle and oncogenic trans-formation [24]MSH6 is a DNAmismatch repair protein thatparticipates in the recognition of mismatched nucleotidesprior to their repair [25] Mutations in this gene have beenassociated with hereditary nonpolyposis colon cancer andendometrial cancer [26] The ZEB1 gene encodes a proteinwhich belongs to a large family of zinc finger transcriptionalfactors [27] Identification of the role of miRNA in theregulation of the gene and the gene set expression is animportant problem Thus we have examined the presence ofmiR-1279 target sites in paralogous genes for PTPN12 MSH6and ZEB1

MiRNA sites are localized within the 51015840UTR CDS and31015840UTR of mRNA [28] Indeed about half of all miRNAbinding sites are located in the protein-coding region [29]suggesting that studies should pay much attention to thesesites The nucleotide placement of these binding sites codingthe amino acid sequencemay raise important questions aboutbinding sites localized in the CDSs

To reduce the number of false positives target sitesmay be validated by conservation of sites across speciesTo verify predicted sites by RNAHybrid we have examinedconservation of these sites across orthologous genes ofdifferent organisms Significant sites in human genes havebeen investigated in PTPN12 MSH6 and ZEB1 orthologuesInvestigating these problems is necessary to establish the roleof miRNA in the regulation of single gene and gene setsexpressions

In present work we analysed the conservation of miR-1279 miR-548m miR-548j and miR-548d-5p binding sitesinorthologues and paralogues of the PTPN12 MSH6 andZEB1 genes to verify these interactions

2 Materials and Methods

Investigation objects were mRNAs of PTPN12 MSH6and ZEB1 human genes their orthologs and paralogs(Supplementary Tables S1 S2 in Supplementary Materialavailable online at httpdxdoiorg1011552013902467) inAnolis carolinensis (Aca) Ailuropoda melanoleuca (Ame)Bos taurus (Bta) Cricetulus griseus (Cgr) Cavia porcel-lus (Cpo) Callithrix jacchus (Cja) Canis lupus familiaris(Clf) Danio rerio (Dre) Equus caballus (Eca) Gallus gal-lus (Gga) Homo sapiens (Hsa) Loxodonta africana (Laf)Macaca mulatta (Mml) Monodelphis domestica (Mdo)Meleagris gallopavo (Mga) Mus musculus (Mmu) Nomascusleucogenys (Nle) Ornithorhynchus anatinus (Oan) Oryctola-gus cuniculus (Ocu) Oreochromis niloticus (Oni) Otolemurgarnettii (Oga) Pan paniscus (Ppa) Papio anubis (Pan)Pan troglodytes (Ptr) Pongo abelii (Pab) Rattus norvegi-cus (Rno) Saimiri boliviensis (Sbo) Sarcophilus harrisii(Sha) Sus scrofa (Ssc) Taeniopygia guttata (Tgu) Xeno-pus laevis (Xla) and Xenopus tropicalis (Xtr) Nucleotideand amino acid sequences were accessed from GenBank

(httpwwwncbinlmnihgov) Nucleotide sequence of hsa-miR-1279 hsa-miR-548m and hsa-miR-548j were receivedfrom miRBase (httpwwwmirbaseorg)

Free energy (Δ119866) position of potential binding sites andinteraction schemes were calculated by RNAhybrid 21 soft-ware (httpbibiservtechfakuni-bielefelddernahybrid)The E-RNAhybrid software (httpsitesgooglecomsitemalaheeneesoftware) was used to compute the ratio ofΔ119866Δ119866

119898and 119875value Δ119866Δ119866

119898value equalled 75 and

more was used as comparative criterion of miRNA andmRNA interaction force Δ119866Δ119866

119898value shows degree of

complementarity of each miRNA site in target gene Δ119866value and its standard deviation were used to determinesignificance level of miRNA binding sites with mRNAInteraction energies (Δ119866

119898) of hsa-miR-1279 hsa-miR-

548m hsa-miR-548 j and hsa-miR-548 d-5 p with theirperfectly complementary sequence were calculated andequaled minus118 kJmoL minus139 kJmoL minus161 kJmoL andminus148 kJmoL consequently At first miRNA binding siteswere found for human mRNAs and then they were revealedin homologous and paralogous genes of studied animalsDiagrams of nucleotide and protein variabilitywere producedby WebLogo software (httpweblogothreeplusonecom)

3 Results

31 Features of hsa-miR-1279 Binding Sites in mRNAs ofParalogous and Orthologous PTPN12 Genes To investigatethe role ofmiR-1279 in the regulation of tyrosine phosphatasefamily genes we attempted to elucidate target binding sitesamong them We identified the PTPN12 gene as a reliabletarget of hsa-miR-1279 with binding energy of minus1021 kJmoLand a Δ119866Δ119866

119898value of 865 hsa-miR-1279 consists of

17 nucleotides 15 of these nucleotides are perfectly comple-mentary to the binding site of human PTPN12 mRNA WeconsideredmiRNAbinding siteswithΔ119866Δ119866

119898values greater

than 75 All sites with lower values were determined to beweak sites and were not consideredThe hybridization energyof miR-1279 with the mRNA of tyrosine phosphatase familygenes except that of PTPN12 varied from minus677 kJmoL tominus895 kJmoL Corresponding Δ119866Δ119866

119898values ranged from

574 to 750 Thus only PTPN12 was considered Thenucleotide sequence of the binding site in PTPN12 encodedthe TKEQYE hexapeptide which was not a conserved aminoacid sequence in any other tyrosine phosphatase familyConsequentlymiR-1279may efficiently regulate onlyPTPN12gene expression

Furthermore we investigated the conservation of themiR-1279 binding site in orthologous genes The humanPTPN12 gene has 23 orthologues in different animals Fordetermination of the conservation of the relevant site weverified the nucleotide sequence of the binding site Thenucleotide sequences of these sites in the mRNA of theinvestigated genes are presented in Table 1 Sequences of themRNAPTPN12 binding sites in all animalswere homologousand nucleotides in first and second positions of the codonswere conserved (Figure 1(a)) ACGAAGGAGCAGUAUGAAoligonucleotide of the interaction site in the CDS encoded the





Position inCDS nt

Δ119866Δ119866m








119898values






210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 39984005998400

(a)

20(s

ites)

N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C

(b)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 39984005998400

(c)

2

0(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(d)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(e)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(f)

2

1

0

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 39984005998400

(g)

210(

sites

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(h)

2

0

3

1(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(i)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(j)

3210

(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12

(k)

210

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(l)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

(m)


its high Δ119866Δ119866119898








119898values



















812 894812 894812 894602 894818 894629 894929 894809 894806 894578 894758 894

1019 876812 883809 894998 876815 894



Position inCDS nt

Δ119866Δ119866m




Position inCDS nt

Δ119866Δ119866m





HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu



6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m



Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m



Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645


Mdo 1950 594Mga 1872 606




Position inCDS nt

Δ119866Δ119866m



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Position inCDS nt

Δ119866Δ119866m








119898values






210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 39984005998400

(a)

20(s

ites)

N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C

(b)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 39984005998400

(c)

2

0(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(d)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(e)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(f)

2

1

0

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 39984005998400

(g)

210(

sites

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(h)

2

0

3

1(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(i)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(j)

3210

(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12

(k)

210

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(l)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

(m)


its high Δ119866Δ119866119898








119898values



















812 894812 894812 894602 894818 894629 894929 894809 894806 894578 894758 894

1019 876812 883809 894998 876815 894



Position inCDS nt

Δ119866Δ119866m




Position inCDS nt

Δ119866Δ119866m





HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu



6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m



Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m



Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645


Mdo 1950 594Mga 1872 606




Position inCDS nt

Δ119866Δ119866m



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 39984005998400

(a)

20(s

ites)

N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 C

(b)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 39984005998400

(c)

2

0(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(d)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(e)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(f)

2

1

0

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 39984005998400

(g)

210(

sites

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(h)

2

0

3

1(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(i)

210(s

ites)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2439984005998400

(j)

3210

(site

s)

N C1 2 3 4 5 6 7 8 9 10 11 12

(k)

210

(site

s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39984005998400

(l)

20(s

ites)

N C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

(m)


its high Δ119866Δ119866119898








119898values



















812 894812 894812 894602 894818 894629 894929 894809 894806 894578 894758 894

1019 876812 883809 894998 876815 894



Position inCDS nt

Δ119866Δ119866m




Position inCDS nt

Δ119866Δ119866m





HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu



6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m



Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m



Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645


Mdo 1950 594Mga 1872 606




Position inCDS nt

Δ119866Δ119866m



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119898values



















812 894812 894812 894602 894818 894629 894929 894809 894806 894578 894758 894

1019 876812 883809 894998 876815 894



Position inCDS nt

Δ119866Δ119866m




Position inCDS nt

Δ119866Δ119866m





HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu



6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m



Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m



Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645


Mdo 1950 594Mga 1872 606




Position inCDS nt

Δ119866Δ119866m



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




812 894812 894812 894602 894818 894629 894929 894809 894806 894578 894758 894

1019 876812 883809 894998 876815 894



Position inCDS nt

Δ119866Δ119866m




Position inCDS nt

Δ119866Δ119866m





HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu



6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m



Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m



Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645


Mdo 1950 594Mga 1872 606




Position inCDS nt

Δ119866Δ119866m



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



HsaAme

CjaPanPpaPtr

SboBtaClfPabEcaLaf

Ocu



6521010101010101010101010101009

620941938

1010989

Position inCDS nt

803

803803

803803803803803803803

81803795

Δ119866Δ119866m



Ame

2263 7632263 7632263 7632263 7632195 7631905 7631878 7512336 7632201 752243 8082263 751



Position inCDS nt

Δ119866Δ119866m



Pab 1521 659Ppa 1875 659Sbo 1875 659Aca 1881 645


Mdo 1950 594Mga 1872 606




Position inCDS nt

Δ119866Δ119866m



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



miR-1279


miR-1279


miR-1279


miR-548j


miR-548m


miR-548d-5p


mRNA PTPN12

mRNA MSH6

mRNA ZEB1

mRNA PTPN12

mRNA PTPN12

mRNA PTPN12

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400



4 Discussion












5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusion


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ptpn12 msh6 ,and - hindawi publishing...

Documents