IDENTIFICATION OF IN VIVO MRNA LIGANDS OF HNRNP A2

描述 翻译后基因中重要的反式作用因子的研究 监管一直受到识别这些mrna的困难的限制。 体内的配体。我们之前已经确定hnRNP A2是特定的 在体内结合GLUT-1和血管内皮生长因子mRNA，最近的数据表明它 在体内也与硫氧还蛋白还原酶mRNA结合。所有的基因也一样 作为硫氧还蛋白还原酶上的hnRNP A2，将提供对它们作用的洞察 在恶变过程中。一个更一般的层面，识别 HnRNP A2的mRNA配体将促进对mRNA-hnRNP的理解 相互作用，进而有助于解决绑定中的差异 不同hnRNPs对相似顺式作用信使核糖核酸元件的亲和力。 鉴定与hnRNP A2、293细胞多聚体特异结合的mRNAs 过表达标记hnRNP A2、hnRNP A1或细胞质 定位突变体，免疫沉淀并作为探针用于筛选 293细胞株的cDNA文库。DNA测序用于鉴定阳性 克隆和未知克隆是完全测序的。进一步的分析将 检测硫氧还蛋白还原酶的周转率以及其他 在存在过表达的hnRNP A2的情况下识别的消息， 融合、细胞生长和缺氧。此消息的结果，在 HnRNP A2过表达的存在、融合、细胞生长和缺氧。 这项研究的结果将有助于阐明hnRNP A2对 硫氧还蛋白还原酶的半衰期，并可为深入了解 HnRNP A2和硫氧还蛋白在多种肿瘤中的作用 有可能为化疗提供一个场所，或者直接 在hnRNP A2或其结合的mRNA的蛋白质产物的下游。

DESCRIPTION The study of trans-acting factors important in post-translational gene regulation has been limited by the difficulty of identifying these mRNA ligands in vivo. We have previously identified hnRNP A2 as specifically binding GLUT-1 and VEGF mRNA in vivo, and recent data indicates that it also binds to thioredoxin reductase mRNA in vivo. All of the genes as well as hnRNP A2 on thioredoxin reductase will provide insight into their roles in malignant transformation. One a more general level, identification of the mRNA ligand of hnRNP A2 will improve the understanding of mRNA-hnRNP interactions which in turn could help to resolve the difference in binding affinities of different hnRNPs to similar cis-acting mRNA elements. To identify mRNAs specifically bound by hnRNP A2, polysomes from 293 cells over-expression FLAG tagged hnRNP A2, hnRNP A1, or cytoplasmically localizing mutants, are immunprecipitated and used as probe to screen a 293 cell line cDNA library. DNA sequencing is used to identify positive clones and unknown clones are fully sequenced. Further analysis will examine the turnover rates of thioredoxin reductase, as well as other identified messages, in the presence of over-expressed hnRNP A2, confluence, cell growth and hypoxia. The results of this messages, in the presence of over-expressed hnRNP A2, confluence, cell growth and hypoxia. The results of this study will help elucidate the impact of hnRNP A2 on the half-life of thioredoxin reductase and could provide insight into the role of hnRNP A2 as well as thioredoxin in various neoplasms, as well as potentially provide a site for chemotherapeutic treatment, either direct at hnRNP A2 or downstream at the protein products of the mRNA it binds.