【作者】 聂代邦；

【导师】 张永亮；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 RNA编辑是近年来分子生物学领域研究的热点。RNA编辑酶ADAR1通过脱氨作用,使RNA特异位点腺嘌呤核苷变为次黄嘌呤核苷,即A-I转换。而在核苷酸中次黄嘌呤核苷的功能与鸟嘌呤(G)的功能是一致的,因而A-I的转换实际上就是A-G的转换。A-I RNA编辑可以改变RNA的序列和二级结构,影响最终的蛋白和调节性RNA,还可以改变mRNA的翻译、pre-mRNA的剪接和与RNA结构相关的事件。ADAR1广泛分布于动物体内各组织,参与胚胎发育、中枢神经系统的神经递质传递、造血系统的发育和机体的免疫等生物学过程。许多维持生命活动的重要功能蛋白质都受到A-I RNA编辑的调控,使其结构和功能以及信号传导通路受到影响。因此,深入理解RNA编辑的机制和靶标将会揭示一些疾病的发病机理,乃至为治疗提供新的思路。目前,ADAR1在造血特别是造血前体细胞中的功能已经比较清楚,但是还不清楚它在T细胞发育中是否具有一定的功能。本研究首先建立T细胞特异ADAR1敲除小鼠模型,PCR分析发现ADAR1敲除特异发生在T细胞;通过对外周血、脾脏、淋巴结和胸腺细胞进行流式分析发现ADAR1基因敲除导致成熟T细胞大量减少;组织化学分析发现T细胞特异ADAR1敲除小鼠胸腺发育异常,与正常小鼠相比大量细胞缺失;进一步进行流式分析发现ADAR1敲除引起DN4前体细胞的死亡,并阻止其在胸腺中的成熟;同时,ADAR1敲除导致TCRβ+细胞大量减少,尽管目前通过重组PCR没有发现ADAR1敲除引起了TCRβ基因座重组的变化,但是这一问题还需要我们进一步研究。此外,Q-PCR结果显示敲除ADAR1激活干扰素信号通路,上调部分干扰素诱导基因的表达。本研究首次发现ADAR1在T细胞发育中具有十分重要的功能,对前体细胞的存活和进一步发育起着重要的作用,提示ADAR1介导的A-I RNA编辑可能参与免疫稳态的调节,它是克隆选择中决定T细胞发育命运的一个尚未被发现的调控因子。更多还原

【Abstract】 The mechanisms accounting for self-tolerance by the immune system and clonal selection of functional lymphocytes that only react to alien antigens are of central concern to immunology. Lymphoid progenitors arising from bone marrow stem cells migrate into thymus where they interact with the cortical and medullar thymic epithelial cells to differentiate into mature T cells. From the progenitor to mature T cell stage, thymocytes have been phenotypically characterized to distinct stages featuring the differentiation antigen expression of CD4 and CD8, defined as double negative (DN), double positive (DP) and single positive (SP) cells. Based on the transient expression of CD44 and CD25, DN cells are further categorized into DN1, 2, 3 and 4 subpopulations. T cell development is an ordered process through multiple differentiation steps, and T cell receptor (TCR) expression is one of the most critical events in the differentiation and maturation processes. TCRβrecombination begins at the DN2 stage and continues to DN3 stage. Functional TCRβis expressed on the surface of DN3 cells, where it associates with pre-TCRαand CD3 elements to form the Pre-TCR which provides signals for proliferation, survival and further maturation. At DP stage, TCRαundergoes V(D)J recombination and both TCRαand TCRβare expressed to form the cell surface TCRα/βreceptors. Functional T cell clones subsequently undergo the positive and negative selection and lineage commitment of CD4 or CD8 SP cells. The mechanism responsible for cell fate during the selection is yet to be fully understood.Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-editing enzyme and plays an essential role in embryonic hematopoiesis. Through its deamination activity, ADAR1 converts adenosine to inosine in RNA molecules (A to I RNA editing); inosine is equivalent to guarnosine in Watson-Crick structure and during protein translation. A-I RNA editing therefore durably modifies sequences of targeted RNAs and can alter proteins that they encode. Proteins coded by edited RNAs, such as GluR-B and 5-HT2cR exhibit dramatically different properties from the genome coded forms. Splicing sites can be generated or eliminated by this A to I conversion. Some microRNA precursors were edited, which consequently modulated the efficacy of mature microRNA genesis or shifts their targets. In addition, ADAR1 has been proposed to participate, through protein-protein interaction, in the processes of RNA transportation, degradation, and regulation of gene expression. Our data has demonstrated an essential role of ADAR1 in T cell development which was associated with TCRβchain expression.ADAR1 plays an important role in host antiviral mechanisms and immunity. Interferons induce the upregulation of ADAR1, thus raising the possibility that ADAR1 functions in host defense mechanisms against viral infection and inflammation. For instance, ADAR1 edits the hepatitis C virus RNA genome and inhibits its replication. Inosine-containing mRNAs increase in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators, including tumor necrosis factor-αand interferon-γ. Furthermore, ADAR1 interacts with nuclear factor 90 (NF90) family proteins. Dysfunction of the A-I RNA editing mechanism can cause human diseases or pathophysiology, Many human diseases related to A-I RNA editing has recently become known.The RNA editing enzyme ADAR1 has been shown to be essential for embryonic development and for the survival of differentiating hematopoietic progenitors. We recently reported that RNA editing activity of ADAR1 was required for the differentiation of adult hematopoietic progenitor cells. ADAR1 is expressed throughout T cell development in the thymus. However, little is understood about its function because of the embryonic lethality of traditional ADAR1-null mutations. In order to explore the function of ADAR1 in T cells, we generated a mouse model in which ADAR1 was specifically deleted in the early stage of T cell differentiation. we found that ADAR1 is required for T cell development at the DN stage. Loss of ADAR1 caused cell death in the progenitors at the DN4 stage and prevented T cell maturation in the thymus. TCRβexpressing cells preferentially diminished in the absence of ADAR1 although the recombination of TCRβloci was shown to be ADAR1-independent. Interruption of IFN signaling was associated with the defect of ADAR1 deficiency. These findings demonstrated an essential role for the RNA editing enzyme ADAR1 supporting in T cell survival after pre-TCR expression and raise the prospect that RNA editing contributes to immunologic homeostasis and is a heretofore unrecognized regulator of T-cell fate during clonal selection.更多还原