Functions of ADARI RNA Editase in Erythropoiesis

RNA编辑通过产生不在基因序列中编码的蛋白质，在某些基因产品的表达中发挥关键作用。一种类型的RNA编辑涉及将腺苷残基转化为肌苷。这种A-to-I RNA编辑是由一个新兴基因家族的多个成员执行的，ADAR(作用于RNA的腺苷脱氨酶)。已在人类和啮齿动物中鉴定出三个不同的ADAR基因家族成员(ADAR1-3)，它们在底物和编辑位点的选择性上显示出显著的差异。对ADAR1缺失突变和嵌合小鼠胚胎的分析表明，大多数胚胎在妊娠中期死亡，红细胞生成系统存在缺陷。结果表明，红系细胞的异常增殖和/或分化是由于目前未知的ADAR1靶基因(S)的RNA编辑不足所致。在这一应用中，我们建议确定在ADAR1缺失突变胚胎中观察到的红系缺陷的分子基础。首先，我们将进行一系列实验，以区分异常红细胞生成是由细胞自主缺陷引起的，还是由胎肝微环境异常引起的。携带新的ADAR1缺失突变等位基因(ADAR1del)和胚胎肝脏特异性ADAR1缺失突变等位基因(ADAR1flx/Alb：AFP-Cre)的小鼠或胚胎将被用于体外红系祖细胞分化实验和宫内移植实验。ADAR1 del/del(-/-)纯合子ES细胞系将用于体外分化实验，也用于嵌合小鼠的形成和组织贡献分析。通过体外培养基因靶向的ES细胞和/或ADAR1缺失的胎儿肝脏而产生的ADAR1缺失的红系细胞将被用于鉴定和克隆对胚胎红系成熟至关重要的ADAR1靶基因(新的A-to-I RNA编辑位点)。从拟议的实验中获得的信息将使我们更好地理解A-to-I RNA编辑的生理意义以及ADAR1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，从而为某些人类红细胞生成障碍的治疗干预提供新的策略。

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.