Functions of ADARI RNA Editase in Erythropoiesis

RNA编辑通过产生基因序列中不编码的蛋白质在某些基因产物的表达中起着关键作用。 一种类型的RNA编辑涉及腺苷残基转化为肌苷。 这种从A到I的RNA编辑是由一个新兴的基因家族阿达尔（作用于RNA的腺苷脱氨酶）的多个成员进行的。 在人类和啮齿类动物中已经鉴定了三个独立的阿达尔基因家族成员（ADAR 1 -3），其在底物和编辑位点选择性方面显示出显著差异。 阶段ADAR 1无效突变和嵌合小鼠胚胎的分析显示，大多数胚胎在妊娠中期死亡，红细胞生成系统缺陷。结果表明，红系细胞的异常增殖和/或分化是由目前未知的ADAR 1靶基因的RNA的欠编辑引起的。在本申请中，我们建议确定ADAR 1无效突变胚胎观察到的红细胞生成缺陷的分子基础。首先，我们将进行一系列的实验，以区分是否异常红细胞生成是由细胞自主缺陷，或异常的胎儿肝脏微环境。携带新的ADAR 1无效突变等位基因（ADAR 1del）和最近通过Cre-loxP重组系统建立的胚胎肝特异性ADAR 1无效突变等位基因（ADAR 1flox/Alb：AFP-Cre）的小鼠或胚胎将用于红系祖细胞分化的体外测定和子宫内移植实验。 ADAR 1 del/del（-/-）纯合ES细胞系将用于体外分化实验，也用于嵌合小鼠形成和组织贡献分析。 通过基因靶向ES细胞和/或ADAR 1缺失胎肝的体外培养产生的ADAR 1缺失红系细胞随后将用于鉴定和克隆对胚胎红系成熟至关重要的ADAR 1靶基因（新的A至I RNA编辑位点）。从拟议的实验中获得的信息将使我们能够更好地了解A-to-I RNA编辑的生理意义以及ADAR 1在发育过程中调节红细胞生成中所起的作用。 我们的研究可能会揭示关键信息，导致某些人类红细胞生成障碍的治疗干预的新策略。

RNA editing plays a critical role in the expression of certain gene products by generating proteins not encoded in the gene sequence. One type of RNA editing involves the conversion of adenosine residues into inosine. This A-to-I RNA editing is carried out by multiple members of an emerging gene family, ADAR (adenosine deaminases acting on RNA). Three separate ADAR gene family members (ADAR1-3) which display significant differences in their substrate and editing site selectivity have been identified in humans and rodents. Analysis of staged ADAR1 null mutant and chimeric mouse embryos revealed that most embryos died at midgestation stage with defects in the erythropoiesis system. The results indicate that abnormal proliferation and/or differentiation of erythroid cells is caused by underediting of the RNA of currently unknown ADAR1 target gene(s). In this application, we propose to determine the molecular basis of erythrogenic defects observed with ADAR1 null mutant embryos. First, we will conduct a series of experiments to distinguish whether abnormal erythropoiesis is caused by cell-autonomous defects, or abnormalities in the fetal liver microenvironment. Mice or embryos harboring a new ADAR1 null mutation allele (ADAR1del) and an embryonic liver-specific ADAR1 null mutation allele (ADAR1flox/Alb:AFP-Cre) established recently by the Cre- loxP recombination system will be used for in vitro assay of erythroid progenitor cell differentiation and in utero transplantation experiments. ADAR1 del/del (-/-) homozygous ES cell lines will be used for in vitro differentiation experiments and also for chimeric mice formation and tissue contribution analysis. ADAR1 null erythroid cells generated by in vitro culture of gene targeted ES cells and/or ADAR1 null fetal livers will then be used for identification and cloning of ADAR1 target genes (new A-to-I RNA editing sites) critical for embryonic erythroid maturation. The information gained from the proposed experiments will allow us to better understand the physiological significance of A-to-I RNA editing and the role played by ADAR1 in the regulation of erythropoiesis during development. Our research may reveal critical information leading to new strategies for therapeutic intervention for certain human dyserythropoietic disorders.