斑马鱼ints12的CRISPR/Cas9敲除及其对UsnRNA剪接的调控  

CRISPR/CAS9-MEDIATED KNOCKOUT OF INTS12 IN ZEBRAFISH AND THE REGULATION OF INTS12 ON USNRNA PROCESSING

作　　者：黄思雨 何牡丹 王厚鹏[1] 孙永华[1,2] HUANG Si-Yu;HE Mu-Dan;WANG Hou-Peng;SUN Yong-Hua(State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China;College of Advanced Agricultural Sciences,University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院大学现代农业科学学院,北京100049

出　　处：《水生生物学报》2019年第1期1-8,共8页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(31671501和31721005)资助~~

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.

关 键 词：ints12 CRISPR/Cas9 UsnRNA mRNA加工 

分 类 号：Q344.1[生物学—遗传学]