Genetic Analysis of EBV's Immortalizing Genes

描述（由申请人提供）：我们最近在该资助期内的发现揭示了典型伯基特淋巴瘤（BL）和WP限制性BL对EBV BART miRNA的意外依赖性（Vereide和Sugden，2011; Vereide et al.，提交）。我们还发现EBV的miRNA促进新感染的B细胞的转化（濑户等人，2010; Vereide等人，提交）。我们建议确定由EBV的miRNA调控的mRNA，以驱动肿瘤维持和促进转化。EBV编码25种pre-miRNAs，可产生50种成熟miRNAs。这些miRNAs预测了超过30%的人类mRNAs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的miRNA的调节（Kuzembayeva et al. submitted; Dolken et al. 2010）。因此，有必要开发功能测定法，以允许表征miRNA的假定靶标，以了解由miRNA介导的其表达的细微差异是否具有功能后果。我们已经开发了当miRNA在生理水平表达时依赖于EBV的miRNA的肿瘤维持和转化的功能测定。后一种鉴定是重要的，因为miRNA以剂量依赖性方式起作用，并且EBV的miRNA通常以低水平表达（Pratt et al.，2009年）。我们建议鉴定调节给定表型的EBV miRNAs的最小子集，然后开发匹配的细胞对，这些细胞从EBV质粒表达和不表达这些miRNAs。从这些细胞对免疫沉淀的RISC中的mRNA将通过深度测序、生物信息学分选来鉴定和计数，并且在报告基因测定中测试它们的3 'UTR以用于特定病毒miRNA的调节。我们已经成功地使用了所有这些测定法来鉴定胱天蛋白酶3作为典型BL中BART miRNA的靶标（Vereide等人提交）。然后将对通过这组测定法发现的mRNA进行功能性测试，以确定其对维持淋巴瘤和转化原代B细胞的潜在贡献。

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.