奶牛乳腺CSN1S1基因线状与环状可变剪接体鉴定分析及功能探讨
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摘要
CSN1S1基因是调控奶牛乳汁中酪蛋白合成的一个重要基因。为鉴定分析奶牛CSN1S1基因可变剪接体种类及分布,验证其环状结构及表达能力,本研究根据牛CSN1S1基因m RNA序列(NM_181029.2)和不同外显子序列分别设计线状及环状引物,用PCR和RT-PCR技术克隆CSN1S1基因可变剪接体,筛选分析不同线状和环状可变剪接体及特征。结果发现:40个阳性克隆中,除全长CSN1S1 mRNA外,共获得12种CSN1S1线状可变剪接体,通过NCBI数据库比对分析确定了其序列组成及所占比重;检测到3种具有完整反向拼接的序列,同时发现多种滚环式结构,进一步确认环形RNA的存在;为验证环状可变剪接体能否表达,实验构建一种过表达载体pEGFP-CSN1S1-12～16 (包含CSN1S1基因12～16号外显子序列),转染293T细胞,48 h后在荧光显微镜下发现有绿色荧光效应,证明其具有翻译潜能。本研究成功筛选出不同线状与环状可变剪接体,并对其表达能力进行验证,为探讨如何提高牛奶酪蛋白合成提供新的依据。
The CSN1 S1 gene is an important gene in the regulation of casein synthesis in milk. We aimed to identify and analyze the distribution and content of alternative spliceosomes of CSN1 S1 gene, and to verify its ring structure and expression ability. In this study designed primers based on the CSN1 S1 mRNA sequence(NM_181029.2). PCR was used to clone and screen different linear alternative spliceosomes. At the same time, the circular primers were designed according to different exons' sequences of this gene. RT-PCR was used to clone and verify the different circular sequences and their characteristics. As a result, among the 40 positive clones, 12 CSN1 S1 linear alternative spliceosomes were obtained in addition to full length of CSN1 S1 cDNA. The sequence composition and its proportion were determined by NCBI database alignment analysis. Three sequences with complete exon splicing were identified in the sequencing of circular alternative spliceosomes. The discovery of the rolling ring structure further confirmed the presence of circular RNA. In order to verify whether circular alternative spliceosomes whic h we have found out have the ability to express, a kind of expression plasmid of circular alternative spliceosome pEGFP-CSN1 S1-12～16 was constructed. Expression plasmid pEGFP-CSN1 S1-12～16 consisted of exon 12～16 of the CSN1 S1 gene. After expression plasmid transformed into 293 T cells, we found a green fluorescent effect during 48 h observation by fluorescence microscopy. This phenomenon proved that it has translation potential. In this experiment, different linear and circular alternative spliceosomes were successfully screened, and their expression ability was validated, which provided the basis for improving the content of milk casein.
The CSN1 S1 gene is an important gene in the regulation of casein synthesis in milk. We aimed to identify and analyze the distribution and content of alternative spliceosomes of CSN1 S1 gene, and to verify its ring structure and expression ability. In this study designed primers based on the CSN1 S1 mRNA sequence(NM_181029.2). PCR was used to clone and screen different linear alternative spliceosomes. At the same time, the circular primers were designed according to different exons' sequences of this gene. RT-PCR was used to clone and verify the different circular sequences and their characteristics. As a result, among the 40 positive clones, 12 CSN1 S1 linear alternative spliceosomes were obtained in addition to full length of CSN1 S1 cDNA. The sequence composition and its proportion were determined by NCBI database alignment analysis. Three sequences with complete exon splicing were identified in the sequencing of circular alternative spliceosomes. The discovery of the rolling ring structure further confirmed the presence of circular RNA. In order to verify whether circular alternative spliceosomes whic h we have found out have the ability to express, a kind of expression plasmid of circular alternative spliceosome pEGFP-CSN1 S1-12～16 was constructed. Expression plasmid pEGFP-CSN1 S1-12～16 consisted of exon 12～16 of the CSN1 S1 gene. After expression plasmid transformed into 293 T cells, we found a green fluorescent effect during 48 h observation by fluorescence microscopy. This phenomenon proved that it has translation potential. In this experiment, different linear and circular alternative spliceosomes were successfully screened, and their expression ability was validated, which provided the basis for improving the content of milk casein.
引文
Artero R.,Prokop A.,P aricio N.,Begemann G.,Pueyo I.,Mlodzik M.,Perez-Alonso M.,and Baylies M.K.,1998,The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2,Dev.Biol.,195(2):131-143
Begemann G.,Paricio N.,Artero R.,Kiss I.,Perez-Alonso M.,and Mlodzik M.,1997,Muscleblind,a gene required for photorecep tor differentiation in Drosophila,encodes novel nuclear Cys3His-type zinc-finger-containing proteins,Development,124(21):4321-4331
Blencowe B.J.,2006,Alternative splicing:new insights from global analyses,Cell,126(1):37-47
Capel B.,Swain A.,Nicolis S.,Hacker A.,Walter M.,Koopman P.,Goodfellow P.,and Lovell-Badge R.,1993,Circular transcripts of the testisdetermining gene Sry in adult mouse testis,Cell,73(5):1019-1030
Cartegni L.,Chew S.L.,and Krainer A.R.,2002,Listening to silence and understanding nonsense:exonic mutations that affect splicing,Nat.Rev.Genet,3(4):285-298
Chianese L.,Quarto M.,Pizzolongo F.,Calabrese M.G.,Caira S.,Mauriello R.,Pascale S.D.,and Addeo F.,2009,Occurrence of genetic poly-morphism at theαs1-casein locus in Mediterranean water buffalo milk,International Dairy Journal,19:181-189
Erhardt G.,Shuiep E.S.,Lisson M.,Weimann C.,Wang Z.X.,El Zubeir I.E.M.,and Pauciullo A.,2016,Alpha S1-casein polymorphisms in camel(Camelus dromedarius)and descriptions of biological active peptides and allergenic epitopes,Trop.Anim.Health Prod.,48(5):879-887
Gao Y.,Wang J.F.,Zheng Y.,Zhang J.Y.,Chen S.,and Zhao F.Q.,2015,Comprehensive identification of internal structure and alternative splicing events in circular RNAs,Nat.Commun.,7:12060
Ghigna C.,Giordano S.,Shen H.,Benvenuto F.,Castiglioni F.,Comoglio P.M.,Green M.R.,Riva S.,and Biamonti G.,2005,Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene,Mol.Cell,20(6):881-890
Guo J.U.,Agarwal V.,Guo H.,and Bartel D.P.,2014,Expanded identification and characterization of mammalian circular RNAs,Genome Biol.,15(7):409
Hansen T.B.,Jensen T.I.,Clausen B.H.,Bramsen J.B.,Finsen B.,Damgaard C.K.,and Kjems J.,2013,Natural RNA circles function as efficient microRNA sponges,Nature,495(7441):384-388
Jeck W.R.,Sorrentino J.A.,Wang K.,Slevin M.K.,Burd C.E.,Liu J.,Marzluff W.F.,and Sharpless N.E.,2013,Circular RNAs are abundant,conserved,and associated with ALUrepeats,RNA,19(2):141-157
Katz Y.,Wang E.T.,Airoldi E.M.,and Burge C.B.,2010,Analysis and design of RNA sequencing experiments for identifying isoform regulation,Nat.Methods,7(12):1009-1015
Koczan D.,Hobom G.,and Sevfert H.M.,1991,Genomic organization of the bovine alpha-s1 casein gen,Nueleie.Acids.Res.,19(20):5591-5596
Legnini I.,Di Timoteo G.,Rossi F.,Morlando M.,Briganti F.,Sthandier O.,Fatica A.,Santini T.,Andronache A.,Wade M.,Laneve P.,Rajewsky N.,and Bozzoni I.,2017,Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis,Mol.Cell,66(1):22-37
Li Z.,Huang C.,Bao C.,Chen L.,Lin M.,Wang X.,Zhong G.,Yu B.,Hu W.,Dai L.,Zhu P.,Chang Z.,Wu Q.,Zhao Y.,Jia Y.,Xu P.,Liu H.,and Shan G.,2015,Exon-intron circular RNAs regulate transcription in the nucleus,Nat.Struct.Mol.Biol.,22(3):256-264
Liu J.,Liu T.,Wang X.,and He A.,2017,Circles reshaping the RNA world:from waste to treasure,Mol.Cancer,16(1):58
Memczak S.,Jens M.,Elefsinioti A.,Torti F.,Krueger J.,Rybak A.,Maier L.,Mackowiak S.D.,Gregersen L.H.,Munschauer M.,Loewer A.,Ziebold U.,Landthaler M.,Kocks C.,le Noble F.,and Rajew sky N.,2013,Circular RNAs are a large class of animal RNAs with regulatory potency,Nature,495(7441):333-338
Nagao M.,Maki M.,Sasaki R.,and Chiba H.,1984,Isolation and sequence analysis of bovineαs1-casein cDNA clone,Agric.Biol.Chem.,48(6):1663-166
Noce A.,Pazzola M.,Dettori M.L.,Amills M.,CastellóA.,Cecchinato A.,Bittante G.,and Vacca G.M.,2016,Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep,Anim.Genet.,47(6):717-726
Pamudurti N.R.,Bartok O.,Jens M.,Ashwal-Fluss R.,Stottmeister C.,Ruhe L.,Hanan M.,Wyler E.,Perez-Hernandez D.,Ramberger E.,Shenzis S.,Samson M.,Dittmar G.,Landthaler M.,Chekulaeva M.,Rajewsky N.,and Kadener S.,2017,Translation of CircRNAs,Mol.Cell,66(1):9-21
Pan Q.,Shai O.,Lee L.J.,Frey B.J.,and Blencowe B.J.,2008,Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing,Nat.Genet.,40(12):1413-1415
Pauciullo A.,and Erhardt G.,2015,Molecular characterization of the llamas(lama glama)casein cluster genes transcripts(CS-N1S1,CSN2,CSN1S2,CSN3)and regulatory regions,PLoSOne,10(4):e0124963
Pauciullo A.,Gauly M.,Cosenza G.,Wagner H.,and Erhardt G.,2017,Lama glamaαS1-casein:Identification of new polymorphisms in the CSN1S1 gene,J.Dairy Sci.,100(2):1282-1289
Pettigrew C.A.,and Brown M.A.,2008,Pre-mRNA splicing aberrations and cancer,Front in Biosci.,13:1090-1105
Rizzetto M.,Canese M.G.,Arico S.,Crivelli O.,Trepo C.,Bonino F.,and Verme G.,1977,Immunofluo-rescence detection of new antigen-antibody system(δ/anti-δ)associated to hepatitis Bvirus in liver and in serum of HBs Ag carriers,Gut.,18(12):997-1003
Rybak-Wolf A.,Stottmeister C.,Glaar P.,Jens M.,Pino N.,Giusti S.,Hanan M.,Behm M.,Bartok O.,Ashwal-Fluss R.,Herzog M.,Schreyer L.,Papavasileiou P.,Ivanov A.,魻hman M.,Refojo D.,Kadener S.,and Rajewsky N.,2015,Circular RNAs in the mammalian brain are highly abundant,conserved,and dynamically expressed,Mol.Cell,58(5):870-885
Salzman J.,Chen R.E.,Olsen M.N.,Wang P.L.,and Brown P.O.,2013,Cell-type specific features of circular RNA expression,PLoS Genetics,9(9):e1003777
Salzman J.,Gawad C.,Wang P.L.,Lacayo N.,and Brown P.O.,2012,Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types,Plo SOne,7(2):e30733
Steven P.B.,and Julia S.,2016,Circular RNAs:analysis,expression and potential functions,Development,143:1838-1847
Tatomer D.C.,and Wilusz J.E.,2017,An unchartered journey for ribosomes:circumnavigating circular RNAs to produce proteins,Mol.Cell,66(1):1-2
Tsiplakou E.,Flemetakis E.,Kouri E.D.,Karalias G.,Sotirakoglou K.,and Zervas G.,2016,The effect of long-term under-and overfeeding on the expression of six major milk proteins'genes in the mammary tissue of goats,J.Anim.Physiol.Anim.Nutr.,100(3):422-430
Winston K.,Veronica G.,Sivaraman N.,Robert C.,Patrick O.B.,and Charles G.,2016,Dynamic ASXL1 exon skipping and alternative circular splicing in single human cells,PLoSOne,11(10):e0164085
Yang Y.,Fan X.,Mao M.,Song X.,Wu P.,Zhang Y.,Jin Y.,Yang Y.,Chen L.L.,Wang Y.,Wong C.C.,Xiao X.,and Wang Z.,2017,Extensive translation of circular RNAs driven by N6-methyladenosine,Cell Res.,27(5):626-641
Begemann G.,Paricio N.,Artero R.,Kiss I.,Perez-Alonso M.,and Mlodzik M.,1997,Muscleblind,a gene required for photorecep tor differentiation in Drosophila,encodes novel nuclear Cys3His-type zinc-finger-containing proteins,Development,124(21):4321-4331
Blencowe B.J.,2006,Alternative splicing:new insights from global analyses,Cell,126(1):37-47
Capel B.,Swain A.,Nicolis S.,Hacker A.,Walter M.,Koopman P.,Goodfellow P.,and Lovell-Badge R.,1993,Circular transcripts of the testisdetermining gene Sry in adult mouse testis,Cell,73(5):1019-1030
Cartegni L.,Chew S.L.,and Krainer A.R.,2002,Listening to silence and understanding nonsense:exonic mutations that affect splicing,Nat.Rev.Genet,3(4):285-298
Chianese L.,Quarto M.,Pizzolongo F.,Calabrese M.G.,Caira S.,Mauriello R.,Pascale S.D.,and Addeo F.,2009,Occurrence of genetic poly-morphism at theαs1-casein locus in Mediterranean water buffalo milk,International Dairy Journal,19:181-189
Erhardt G.,Shuiep E.S.,Lisson M.,Weimann C.,Wang Z.X.,El Zubeir I.E.M.,and Pauciullo A.,2016,Alpha S1-casein polymorphisms in camel(Camelus dromedarius)and descriptions of biological active peptides and allergenic epitopes,Trop.Anim.Health Prod.,48(5):879-887
Gao Y.,Wang J.F.,Zheng Y.,Zhang J.Y.,Chen S.,and Zhao F.Q.,2015,Comprehensive identification of internal structure and alternative splicing events in circular RNAs,Nat.Commun.,7:12060
Ghigna C.,Giordano S.,Shen H.,Benvenuto F.,Castiglioni F.,Comoglio P.M.,Green M.R.,Riva S.,and Biamonti G.,2005,Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene,Mol.Cell,20(6):881-890
Guo J.U.,Agarwal V.,Guo H.,and Bartel D.P.,2014,Expanded identification and characterization of mammalian circular RNAs,Genome Biol.,15(7):409
Hansen T.B.,Jensen T.I.,Clausen B.H.,Bramsen J.B.,Finsen B.,Damgaard C.K.,and Kjems J.,2013,Natural RNA circles function as efficient microRNA sponges,Nature,495(7441):384-388
Jeck W.R.,Sorrentino J.A.,Wang K.,Slevin M.K.,Burd C.E.,Liu J.,Marzluff W.F.,and Sharpless N.E.,2013,Circular RNAs are abundant,conserved,and associated with ALUrepeats,RNA,19(2):141-157
Katz Y.,Wang E.T.,Airoldi E.M.,and Burge C.B.,2010,Analysis and design of RNA sequencing experiments for identifying isoform regulation,Nat.Methods,7(12):1009-1015
Koczan D.,Hobom G.,and Sevfert H.M.,1991,Genomic organization of the bovine alpha-s1 casein gen,Nueleie.Acids.Res.,19(20):5591-5596
Legnini I.,Di Timoteo G.,Rossi F.,Morlando M.,Briganti F.,Sthandier O.,Fatica A.,Santini T.,Andronache A.,Wade M.,Laneve P.,Rajewsky N.,and Bozzoni I.,2017,Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis,Mol.Cell,66(1):22-37
Li Z.,Huang C.,Bao C.,Chen L.,Lin M.,Wang X.,Zhong G.,Yu B.,Hu W.,Dai L.,Zhu P.,Chang Z.,Wu Q.,Zhao Y.,Jia Y.,Xu P.,Liu H.,and Shan G.,2015,Exon-intron circular RNAs regulate transcription in the nucleus,Nat.Struct.Mol.Biol.,22(3):256-264
Liu J.,Liu T.,Wang X.,and He A.,2017,Circles reshaping the RNA world:from waste to treasure,Mol.Cancer,16(1):58
Memczak S.,Jens M.,Elefsinioti A.,Torti F.,Krueger J.,Rybak A.,Maier L.,Mackowiak S.D.,Gregersen L.H.,Munschauer M.,Loewer A.,Ziebold U.,Landthaler M.,Kocks C.,le Noble F.,and Rajew sky N.,2013,Circular RNAs are a large class of animal RNAs with regulatory potency,Nature,495(7441):333-338
Nagao M.,Maki M.,Sasaki R.,and Chiba H.,1984,Isolation and sequence analysis of bovineαs1-casein cDNA clone,Agric.Biol.Chem.,48(6):1663-166
Noce A.,Pazzola M.,Dettori M.L.,Amills M.,CastellóA.,Cecchinato A.,Bittante G.,and Vacca G.M.,2016,Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep,Anim.Genet.,47(6):717-726
Pamudurti N.R.,Bartok O.,Jens M.,Ashwal-Fluss R.,Stottmeister C.,Ruhe L.,Hanan M.,Wyler E.,Perez-Hernandez D.,Ramberger E.,Shenzis S.,Samson M.,Dittmar G.,Landthaler M.,Chekulaeva M.,Rajewsky N.,and Kadener S.,2017,Translation of CircRNAs,Mol.Cell,66(1):9-21
Pan Q.,Shai O.,Lee L.J.,Frey B.J.,and Blencowe B.J.,2008,Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing,Nat.Genet.,40(12):1413-1415
Pauciullo A.,and Erhardt G.,2015,Molecular characterization of the llamas(lama glama)casein cluster genes transcripts(CS-N1S1,CSN2,CSN1S2,CSN3)and regulatory regions,PLoSOne,10(4):e0124963
Pauciullo A.,Gauly M.,Cosenza G.,Wagner H.,and Erhardt G.,2017,Lama glamaαS1-casein:Identification of new polymorphisms in the CSN1S1 gene,J.Dairy Sci.,100(2):1282-1289
Pettigrew C.A.,and Brown M.A.,2008,Pre-mRNA splicing aberrations and cancer,Front in Biosci.,13:1090-1105
Rizzetto M.,Canese M.G.,Arico S.,Crivelli O.,Trepo C.,Bonino F.,and Verme G.,1977,Immunofluo-rescence detection of new antigen-antibody system(δ/anti-δ)associated to hepatitis Bvirus in liver and in serum of HBs Ag carriers,Gut.,18(12):997-1003
Rybak-Wolf A.,Stottmeister C.,Glaar P.,Jens M.,Pino N.,Giusti S.,Hanan M.,Behm M.,Bartok O.,Ashwal-Fluss R.,Herzog M.,Schreyer L.,Papavasileiou P.,Ivanov A.,魻hman M.,Refojo D.,Kadener S.,and Rajewsky N.,2015,Circular RNAs in the mammalian brain are highly abundant,conserved,and dynamically expressed,Mol.Cell,58(5):870-885
Salzman J.,Chen R.E.,Olsen M.N.,Wang P.L.,and Brown P.O.,2013,Cell-type specific features of circular RNA expression,PLoS Genetics,9(9):e1003777
Salzman J.,Gawad C.,Wang P.L.,Lacayo N.,and Brown P.O.,2012,Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types,Plo SOne,7(2):e30733
Steven P.B.,and Julia S.,2016,Circular RNAs:analysis,expression and potential functions,Development,143:1838-1847
Tatomer D.C.,and Wilusz J.E.,2017,An unchartered journey for ribosomes:circumnavigating circular RNAs to produce proteins,Mol.Cell,66(1):1-2
Tsiplakou E.,Flemetakis E.,Kouri E.D.,Karalias G.,Sotirakoglou K.,and Zervas G.,2016,The effect of long-term under-and overfeeding on the expression of six major milk proteins'genes in the mammary tissue of goats,J.Anim.Physiol.Anim.Nutr.,100(3):422-430
Winston K.,Veronica G.,Sivaraman N.,Robert C.,Patrick O.B.,and Charles G.,2016,Dynamic ASXL1 exon skipping and alternative circular splicing in single human cells,PLoSOne,11(10):e0164085
Yang Y.,Fan X.,Mao M.,Song X.,Wu P.,Zhang Y.,Jin Y.,Yang Y.,Chen L.L.,Wang Y.,Wong C.C.,Xiao X.,and Wang Z.,2017,Extensive translation of circular RNAs driven by N6-methyladenosine,Cell Res.,27(5):626-641