Functions of ADARI RNA Editase in Erythropoiesis

RNA编辑通过产生基因序列中未编码的蛋白质，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进行的，ADAR（作用于RNA的腺苷脱氨酶）。在人类和啮齿动物中发现了三个独立的ADAR基因家族成员（ADAR1-3），它们在底物和编辑位点选择性上表现出显著差异。对分期ADAR1零突变体和嵌合小鼠胚胎的分析显示，大多数胚胎在妊娠中期死亡，红细胞生成系统存在缺陷。结果表明，红细胞的异常增殖和/或分化是由目前未知的ADAR1靶基因的RNA编辑不足引起的。在这个应用中，我们建议确定在ADAR1零突变胚胎中观察到的红细胞缺陷的分子基础。首先，我们将通过一系列实验来区分红细胞生成异常是由细胞自主缺陷引起的，还是胎儿肝脏微环境异常引起的。新建立的ADAR1零突变等位基因（ADAR1del）和胚胎肝特异性ADAR1零突变等位基因（ADAR1flox/Alb:AFP-Cre）的小鼠或胚胎将用于体外红细胞祖细胞分化实验和子宫移植实验。ADAR1 del/del（-/-）纯合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ES细胞和/或ADAR1缺失的胎儿肝脏产生的ADAR1缺失红细胞将用于鉴定和克隆ADAR1靶基因（新的A-to-I RNA编辑位点），这些基因对胚胎红细胞成熟至关重要。从这些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在发育过程中对红细胞生成的调节中的作用。我们的研究可能会揭示一些关键信息，从而为某些人类促红细胞增生疾病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.