Functions of ADARI RNA Editase in Erythropoiesis

ADARI RNA 编辑酶在红细胞生成中的功能

• 批准号: 6746917
• 负责人: KAZUKO NISHIKURA
• 金额: $ 45.63万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6746917
• 关键词: SDS polyacrylamide gel electrophoresis; adenosine deaminase; affinity chromatography; apoptosis; cell differentiation; cell line; cell proliferation; complementary DNA; embryo /fetus; embryo /fetus transplantation; embryonic stem cell; enzyme activity; erythrocytes; erythropoiesis; gene expression; gene targeting; genetic library; genetically modified animals; in utero transplantation; laboratory mouse; liver cells; mass spectrometry; microarray technology; mutant; posttranscriptional RNA processing

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.

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发挥着关键作用。 一种类型的 RNA 编辑涉及将腺苷残基转化为肌苷。 这种 A 到 I RNA 编辑是由新兴基因家族 ADAR（作用于 RNA 的腺苷脱氨酶）的多个成员进行的。 已在人类和啮齿类动物中鉴定出三个独立的 ADAR 基因家族成员 (ADAR1-3)，其底物和编辑位点选择性表现出显着差异。 对分阶段 ADAR1 无效突变体和嵌合小鼠胚胎的分析表明，大多数胚胎在妊娠中期因红细胞生成系统缺陷而死亡。结果表明，红系细胞的异常增殖和/或分化是由目前未知的 ADAR1 靶基因的 RNA 编辑不足引起的。在此应用中，我们建议确定 ADAR1 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。最近由 CreloxP 重组系统建立的携带新 ADAR1 无效突变等位基因 (ADAR1del) 和胚胎肝脏特异性 ADAR1 无效突变等位基因 (ADAR1flox/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移植实验。 ADAR1 del/del (-/-) 纯合 ES 细胞系将​​用于体外分化实验以及嵌合小鼠形成和组织贡献分析。 通过基因靶向 ES 细胞和/或 ADAR1 缺失胎儿肝脏的体外培养产生的 ADAR1 缺失红系细胞将用于鉴定和克隆 ADAR1 靶基因（新的 A 到 I RNA 编辑位点），这对胚胎红系成熟至关重要。从所提出的实验中获得的信息将使我们能够更好地理解 A-to-I RNA 编辑的生理意义以及 ADAR1 在发育过程中红细胞生成调节中所发挥的作用。 我们的研究可能揭示关键信息，从而为某些人类红细胞生成障碍性疾病的治疗干预提供新策略。