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

描述(申请人提供)：恶性转化代表连续遗传损伤的表型终点，这些损伤改变了癌基因和肿瘤抑制基因网络中的基因功能和基因调节。虽然广泛的研究已经确定了蛋白质编码基因在这些分子网络中的功能，但非编码RNA(NcRNAs)的功能重要性才刚刚开始被揭示。MicroRNAs(MiRNAs)编码一类小的、具有转录后基因调控能力的ncRNAs，通过靶向广泛的mRNAs。我们以前的研究发现了一个重要的miRNA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转录本，产生六个单独的miRNA组分，这些组分具有不同的生物学功能和在肿瘤发生过程中的不同基因调控。这种对多顺反子miRNA成分的上下文相关的差异调控最终可能决定miR17-92的致癌活性。在人类癌症中经常观察到miRNA的生物发生、稳定性和功能的异常调节，但其潜在的分子基础仍不清楚。这在很大程度上是由于缺乏有效的方法来研究癌症相关的miRNA核糖核蛋白(MiRNP)复合体。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定miRNA核糖核蛋白(MiRNP)复合体。我们设计了一个细菌内切酶Csy4，以实现特定的16核苷酸(NT)RNA序列的高亲和力结合和诱导切割。利用这个16-NT的RNA作为标签，我们的目标是在体外和体内分离特定的miRNP复合体。这些拟议的研究将使我们能够识别重要的miRNP复杂成分，这些成分对癌细胞中多顺反子miRNA癌基因的成分有不同的调节作用。更重要的是，我们的技术可以很容易地用于研究与癌症相关的包含其他ncRNA物种的RNP复合体。随着癌症中功能上重要的ncRNAs数量的增加，我们提出的研究将为癌症研究这个在很大程度上尚未被探索的领域带来基本的见解。

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.