斑马鱼ints12的CRISPR/Cas9敲除及其对UsnRNA剪接的调控
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摘要
整合因子(Integrator)复合体通过调控UsnRNAs(U-rich small nuclear RNAs)的转录成熟过程,影响premRNA的内含子剪切,对生物体内形成成熟mRNA有重要作用。但是在脊椎动物中,整合因子复合体的表达调控及其发育功能研究尚十分缺乏。研究利用斑马鱼(Danio rerio)模型,通过CRISPR/Cas9技术构建了ints12(Integrator subunit 12)的基因敲除品系,得到了2种缺失不同碱基的突变品系。在ints12的合子突变体(Zints12)中,通过荧光定量PCR分析,发现UsnRNA的3′box剪切存在缺陷。与同龄的野生型斑马鱼相比,Zints12体型偏小且表现为全雄。进一步研究发现,在Zints12成体中,细胞增殖相关因子出现显著下调表达,ints12自身pre-mRNA的加工也出现内含子滞留,表明ints12对自身mRNA的剪切存在一个"自循环"式的调控模式。研究获得了斑马鱼的ints12基因敲除纯合突变体并发现ints12通过调控UsnRNA的剪切参与斑马鱼的个体生长和发育。
Through regulating the post-transcriptional maturation of UsnRNAs(U-rich small nuclear RNAs), integrator complex has an effect on the intron splicing of pre-mRNA. Therefore, integrator complex plays an important role in the production of mature mRNA. However, the regulation and developmental functions of integrator complex are poorly understood in vertebrates. In this study, zebrafish(Danio rerio) was used to generate the ints12 knockout model by CRISPR/Cas9, and two homozygous lines were generated with different frame-shifts. Observation on the misprocessing of UsnRNA 3′ ends in zygotic mutant of ints12(Zints12) was conducted through real-time quantitative PCR analysis.When compared with the control fish, the overall growth of Zints12 was largely retarded, and the Zints12 population was all-male. Further studies showed that cell proliferation was significantly interfered, and the level of introns retention of ints12 transcripts was significantly increased in Zints12, suggesting an "auto-regulation loop" in the splicing regulation by ints12. Overall, we obtained homozygous mutants of zebrafish ints12 by gene knockout technology and revealed that zebrafish ints12 regulates UsnRNA 3′-box processing to exert various effects on early embryonic development and body growth.
Through regulating the post-transcriptional maturation of UsnRNAs(U-rich small nuclear RNAs), integrator complex has an effect on the intron splicing of pre-mRNA. Therefore, integrator complex plays an important role in the production of mature mRNA. However, the regulation and developmental functions of integrator complex are poorly understood in vertebrates. In this study, zebrafish(Danio rerio) was used to generate the ints12 knockout model by CRISPR/Cas9, and two homozygous lines were generated with different frame-shifts. Observation on the misprocessing of UsnRNA 3′ ends in zygotic mutant of ints12(Zints12) was conducted through real-time quantitative PCR analysis.When compared with the control fish, the overall growth of Zints12 was largely retarded, and the Zints12 population was all-male. Further studies showed that cell proliferation was significantly interfered, and the level of introns retention of ints12 transcripts was significantly increased in Zints12, suggesting an "auto-regulation loop" in the splicing regulation by ints12. Overall, we obtained homozygous mutants of zebrafish ints12 by gene knockout technology and revealed that zebrafish ints12 regulates UsnRNA 3′-box processing to exert various effects on early embryonic development and body growth.
引文
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[26]Sun F,Fu H,Liu Q,et al.Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest[J].FEBSLetters,2008,582(10):1564-1568
[27]Hall P A,Levison D A,Woods A L,et al.Proliferating cell nuclear antigen(PCNA)immunolocalization in paraffin sections:An index of cell proliferation with evidence of deregulated expression in some,neoplasms[J].Journal of Pathology,1990,162(4):285
[28]Hou Y Y,Cao W W,Li L,et al.MicroRNA-519d targetsMKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703[J].Cancer Letters,2011,307(2):182-190
[29]Chen J,Waltenspiel B,Warren W D,et al.Functional analysis of the integrator subunit 12 identifies a microdomain that mediates activation of the Drosophila integrator complex[J].Journal of Biological Chemistry,2013,288(7):4867-4877
[30]Tzung K W,Goto R,Saju J M,et al.Early depletion of primordial germ cells in zebrafish promotes testis formation[J].Stem Cell Reports,2015,4(1):61-73
[31]Rienzo M,Casamassimi A.Integrator complex and transcription regulation:Recent findings and pathophysiology[J].Biochim Biophys Acta,2016,1859(10):1269-1280
[32]Skaar J R,Ferris A L,Wu X,et al.The Integrator complex controls the termination of transcription at diverse classes of gene targets[J].Cell Research,2015,25(3):288-305
[33]Ciruna B,Weidinger G,Knaut H,et al.Production of maternal-zygotic mutant zebrafish by germ-line replacement[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(23):14919
[2]Egloff S,O’Reilly D,Murphy S.Expression of human snRNA genes from beginning to end[J].Biochemical Society Transactions,2008,36(Pt 4):590-594
[3]Matera A G,Terns R M,Terns M P.Non-coding RNAs:lessons from the small nuclear and small nucleolar RNAs[J].Nature Reviews,2007,8(3):209-220
[4]Eliceiri G L,Sayavedra M S.Small RNAs in the nucleus and cytoplasm of hela cells[J].Biochemical&Biophysical Research Communications,1976,72(2):507-512
[5]Kleinschmidt A M,Pederson T.Accurate and efficient 3’processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract[J].Molecular&Cellular Biology,1987,7(9):3131-3137
[6]Uguen P,Murphy S.The 3’ends of human pre-snRNAs are produced by RNA polymerase II CTD-dependent RNA processing[J].EMBO Journal,2003,22(17):4544
[7]de Vegvar H E,Lund E,Dahlberg J E.3’end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters[J].Cell,1986,47(2):259
[8]Hernandez N.Formation of the 3’end of U1 snRNA is directed by a conserved sequence located downstream of the coding region[J].EMBO Journal,1985,4(7):1827-1837
[9]Hernandez N,Weiner A M.Formation of the 3’end of U1snRNA requires compatible snRNA promoter elements[J].Cell,1986,47(2):249-258
[10]Baillat D,Hakimi M A,N??r A M,et al.Integrator,a multiprotein mediator of small nuclear RNA processing,associates with the C-terminal repeat of RNA polymerase II[J].Cell,2005,123(2):265-276
[11]Sylvain E,Anna S S,Martin D,et al.The integrator complex recognizes a new double mark on the RNA polymerase II carboxyl-terminal domain[J].Journal of Biological Chemistry,2010,285(27):20564-20569
[12]Egloff S,Zaborowska J,Laitem C,et al.Ser7 phosphorylation of the CTD recruits the RPAP2 Ser5 phosphatase to snRNA genes[J].Molecular Cell,2012,45(1):111-122
[13]Ezzeddine N,Chen J,Waltenspiel B,et al.A subset of Drosophila integrator proteins is essential for efficient U7snRNA and spliceosomal snRNA 3’-end formation[J].Molecular&Cellular Biology,2011,31(2):328-341
[14]Baillat D,Wagner E J.Integrator:surprisingly diverse functions in gene expression[J].Trends in Biochemical Sciences,2015,40(5):257-264
[15]Chen J,Wagner E J.snRNA 3’end formation:the dawn of the Integrator complex[J].Biochemical Society Transactions,2010,38(4):1082-1087
[16]Peterson R T,Shaw S Y,Peterson T A,et al.Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation[J].Nature Biotechnology,2004,22(5):595-599
[17]Fleming A,Sato M,Goldsmith P.High-throughput in vivo screening for bone anabolic compounds with zebrafish[J].Journal of Biomolecular Screening,2005,10(8):823-831
[18]Tao S,Cai Y,Sampath K.The Integrator subunits function in hematopoiesis by modulating Smad/BMP signaling[J].Development,2009,136(16):2757-2765
[19]Kapp L D,Abrams E W,Marlow F L,et al.The integrator complex subunit 6(Ints6)confines the dorsal organizer in vertebrate embryogenesis[J].PLoS Genetics,2013,9(10):e1003822
[20]Chen J,Ezzeddine N,Waltenspiel B,et al.An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3’-end formation[J].RNA,2012,18(12):2148-2156
[21]Kimmel C B,Ballard W W,Kimmel S R,et al.Stages of embryonic development of the zebrafish[J].Developmental Dynamics,1995,203(3):253-310
[22]Thisse C,Thisse B.High-resolution in situ hybridizationto whole-mount zebrafish embryos[J].Nature Protocols,2008,3(1):59-69
[23]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408
[24]Chang N N,Sun C H,Gao L,et al.Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos[J].Cell Research,2013,23(4):465-472
[25]Zhang F H,Wang H P,Huang S Y,et al.A comparison of the knockout efficiencies of two codon-optimized Cas9coding sequences in zebrafish embryos[J].Hereditas(Beijing),2016,38(2):144-154[张峰华,王厚鹏,黄思雨,等.两种密码子优化的Cas9编码基因在斑马鱼胚胎中基因敲除效率的比较.遗传,2016,38(2):144-154]
[26]Sun F,Fu H,Liu Q,et al.Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest[J].FEBSLetters,2008,582(10):1564-1568
[27]Hall P A,Levison D A,Woods A L,et al.Proliferating cell nuclear antigen(PCNA)immunolocalization in paraffin sections:An index of cell proliferation with evidence of deregulated expression in some,neoplasms[J].Journal of Pathology,1990,162(4):285
[28]Hou Y Y,Cao W W,Li L,et al.MicroRNA-519d targetsMKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703[J].Cancer Letters,2011,307(2):182-190
[29]Chen J,Waltenspiel B,Warren W D,et al.Functional analysis of the integrator subunit 12 identifies a microdomain that mediates activation of the Drosophila integrator complex[J].Journal of Biological Chemistry,2013,288(7):4867-4877
[30]Tzung K W,Goto R,Saju J M,et al.Early depletion of primordial germ cells in zebrafish promotes testis formation[J].Stem Cell Reports,2015,4(1):61-73
[31]Rienzo M,Casamassimi A.Integrator complex and transcription regulation:Recent findings and pathophysiology[J].Biochim Biophys Acta,2016,1859(10):1269-1280
[32]Skaar J R,Ferris A L,Wu X,et al.The Integrator complex controls the termination of transcription at diverse classes of gene targets[J].Cell Research,2015,25(3):288-305
[33]Ciruna B,Weidinger G,Knaut H,et al.Production of maternal-zygotic mutant zebrafish by germ-line replacement[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(23):14919