A new technology to isolate RNP complexes of a polycistronic miRNA oncogene

描述（由申请人提供）：恶性转化代表了改变癌基因和肿瘤抑制网络中基因功能和基因调控的连续遗传病变的表型终点。虽然广泛的研究已经在这些分子网络中描述了蛋白质编码基因的功能，但非编码rna （ncRNAs）的功能重要性才刚刚开始被揭示出来。microRNAs （miRNAs）编码一类小调控ncRNAs，通过靶向广泛的mrna，具有很强的转录后基因调控能力。我们之前的研究发现了一个重要的miRNA致癌基因mir17-92，它促进了b细胞淋巴瘤的发展。mir17-92编码一个多顺反子miRNA转录物，产生6个单独的miRNA组分，它们在肿瘤发生过程中具有不同的生物学功能和不同的基因调控。这种多顺反子miRNA组分的上下文依赖性差异调控最终决定了mir17-92的致癌活性。miRNA生物发生、稳定性和功能的异常调控在人类癌症中经常被观察到，但其潜在的分子基础仍不清楚。这主要是由于缺乏一种有效的方法来研究与癌症相关的miRNA核糖核蛋白（miRNP）复合物。在这里，我们建议开发一种强大的生化策略来分离和表征在肿瘤发生过程中具有重要功能的特异性miRNA核糖核蛋白（miRNP）复合物。我们设计了一种细菌内切酶Csy4，以实现高亲和力结合和诱导切割特定的16核苷酸（nt） RNA序列。使用这种16-nt RNA作为标签，我们的目标是在体外和体内分离特异性miRNP复合物。这些拟议的研究将使我们能够识别重要的miRNP复合物组分，这些复合物组分对癌细胞中多顺反子miRNA癌基因的组分进行差异调节。更重要的是，我们的技术可以很容易地适应于研究含有其他ncRNA物种的癌症相关RNP复合物。随着癌症中功能重要的ncrna数量的增加，我们提出的研究将为癌症研究的一个很大程度上未被探索的领域带来根本性的见解。

DESCRIPTION (provided by applicant): Malignant transformation represents the phenotypic endpoint of successive genetic lesions that alter gene function and gene regulation in oncogene and tumor suppressor networks. While extensive investigations have characterized protein-coding gene functions in these molecular networks, the functional importance of non-coding RNAs (ncRNAs) is just beginning to be revealed. microRNAs (miRNAs) encode a class of small regulatory ncRNAs with great capacity for post-transcriptional gene regulation by targeting a broad range of mRNAs. Our previous studies identified an important miRNA oncogene, mir17-92, that promotes the development of B-cell lymphomas. mir17-92 encodes a polycistronic miRNA transcript that yields six individual miRNA components, which have distinct biological functions and differential gene regulation during tumorigenesis. This context-dependent differential regulation of polycistronic miRNA components could ultimately determine the oncogenic activity of mir17-92. Aberrant regulation of miRNA biogenesis, stability and function has been frequently observed in human cancer, yet the underlying molecular basis still remains unclear. This is largely due to the lack of an effective methodology to study cancer-related miRNA ribonucleoprotein (miRNP) complexes. Here, we propose to develop a powerful biochemical strategy to isolate and characterize specific miRNA ribonucleoprotein (miRNP) complexes with important functions during tumorigenesis. We engineered a bacterial endonuclease, Csy4, to achieve high affinity binding and inducible cleavage of a specific 16-nucleotide (nt) RNA sequence. Using this 16-nt RNA as a tag, we aim to isolate specific miRNP complexes both in vitro and in vivo. These proposed studies will allow us to identify important miRNP complex components that differentially regulate components of a polycistronic miRNA oncogene in cancer cells. More importantly, our technology can be easily adapted to study cancer related RNP complexes containing other ncRNA species. With the increasing number of functionally important ncRNAs in cancer, our proposed studies will bring fundamental insights into an area of cancer research that has been largely unexplored.