Genetic Analysis of EBV's Immortalizing Genes

描述（由申请人提供）：我们在本次资助期间的最新发现揭示了典型伯基特淋巴瘤（BLs）和wp限制性BLs对EBV的BART mirna的意外依赖（Vereide和Sugden, 2011； Vereide等人，提交）。我们还发现EBV的mirna促进新感染b细胞的转化（Seto et al., 2010; Vereide et al.，提交）。我们建议鉴定由EBV的mirna调控的mrna，以驱动肿瘤维持和促进转化。EBV编码25个pre- mirna，可产生50个成熟mirna。这些mirna已经预测了超过30%的人类mrna的种子位点。然而，包括我们自己的研究在内的许多研究表明，绝大多数这些mrna不受EBV mirna的调节（Kuzembayeva等人提交；Dolken等人2010）。因此，有必要开发功能分析，以表征mirna的假定靶标，以便了解mirna介导的表达的细微差异是否具有功能后果。当EBV的mirna在生理水平上表达时，我们已经开发了肿瘤维持和转化的功能分析。后一种鉴定很重要，因为mirna以剂量依赖的方式起作用，而EBV的mirna通常以低水平表达（Pratt等人，2009）。我们建议鉴定EBV调节给定表型的最小mirna亚群，然后从EBV质粒中培养出表达和不表达这些mirna的匹配细胞对。从这些细胞对中免疫沉淀的RISCs中的mrna将通过深度测序和生物信息学分类进行鉴定和枚举，并在报告者试验中检测它们的3' utr，以确定它们是否受特定病毒mirna的调节。我们已经使用所有这些检测方法成功地鉴定了caspase 3作为典型BLs中BART mirna的靶标（Vereide等人提交）。通过这组试验发现的mrna将在功能上进行测试，以确定其对维持淋巴瘤和转化原代b细胞的潜在作用。

DESCRIPTION (provided by applicant): Our recent findings made during this funding period have revealed an unexpected dependence of canonical Burkitt's Lymphomas (BLs) and Wp-restricted BLs on EBV's BART miRNAs (Vereide and Sugden, 2011; Vereide et al., submitted). We have also found that EBV's miRNAs promote transformation of newly infected B-cells (Seto et al., 2010; Vereide et al., submitted). We propose to identify the mRNAs that are regulated by EBV's miRNAs to drive tumor maintenance and promote transformation. EBV encodes 25 pre-miRNAs which can yield 50 mature miRNAs. These miRNAs have predicted seed sites in more than 30% of all human mRNAs. However, many studies, including our own, indicate that the vast majority of these mRNAs are not regulated by EBV's miRNAs (Kuzembayeva et al. submitted; Dolken et al. 2010). It is therefore essential to develop functional assays to allow characterization of presumptive targets of miRNAs in order to learn if the subtle differences in their expression mediated by miRNAs have functional consequences. We have developed functional assays for tumor maintenance and transformation dependent on EBV's miRNAs when the miRNAs are expressed at physiological levels. This latter qualification is important because miRNAs act in a dose-dependent manner, and EBV's miRNAs are often expressed at low levels (Pratt et al., 2009). We propose to identify minimal subsets of EBV's miRNAs that regulate a given phenotype and then develop matched pairs of cells that do and do not express these miRNAs from EBV plasmids. The mRNAs in the RISCs immunoprecipitated from these pairs of cells will be identified and enumerated by deep sequencing, sorted bioinformatically, and their 3'UTRs tested in reporter assays for regulation by specific viral miRNAs. We have used all of these assays successfully in our identification of caspase 3 as a target for BART miRNAs in canonical BLs (Vereide et al. submitted). The mRNAs found by this set of assays will then be tested functionally for potential contributions to maintaining lymphomas and transforming primary B-cells.