DROSHA FUNCTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 MicroRNAs是一种小的非编码调控RNA，与发育和癌症有关。MicroRNA前体由微处理器处理，微处理器是由内切酶DROSHA及其合作伙伴Dgcr8蛋白组成的复合体。DROSHA也是另一个更大的核联合体的一部分，以下称为宏处理器。宏处理器包含几个RNA结合和解离因子以及EWS，EWS是一种与人类肿瘤有关的RNA结合蛋白，但其功能和底物的身份尚不清楚。我打算描述宏处理器复合体的特征，并确定它的底物。为了研究DROSHA？S的功能，我以前曾利用逆转录病毒siRNA生产载体降低Hela细胞中DROSHA蛋白的水平，并通过克隆形成实验发现基因敲除的细胞活性降低。 RNT1是芽殖酵母中唯一的核糖核酸酶III，有几种信使核糖核酸底物。为了确定DROSHA是否也在mRNA加工中发挥作用，我使用微阵列对基因敲除进行了表达分析。总共发现了44组探针，其中17组在DROSHA击倒中增加，27组减少。在减少的27个靶点中，有一个(c-jun)是用两套不同的探针组确定的。正如预期的那样，在所有四个比较中，另一个下降的目标是DROSHA mRNA本身。没有一个靶(DROSHA除外)显示出与任何siRNA结构的同源性，这使得它们不太可能是脱靶效应。 我正在以我大量的初步数据为基础，利用我独特而强大的试剂(亲和力纯化的抗DROSHA抗体和抗DROSHA siRNA击倒构建体)，以及Chait实验室丰富的生化和质谱学资源，来表征DROSHA在巨处理器复合体中的作用。我的具体目标包括： 1.DROSHA底物的分离与鉴定 2.细胞周期内DROSHA？S动力学的测定

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MicroRNAs are small non-coding regulatory RNAs implicated in both development and cancer. MicroRNA precursors are processed by Microprocessor, a complex consisting of the endonuclease drosha and its partner, the DGCR8 protein. Drosha is also part of another larger nuclear complex, hereafter called Macroprocessor. Macroprocessor contains several RNA binding and unwinding factors as well as EWS, an RNA-binding protein involved in human tumors, but its function and the identities of its substrates are unknown. I intend to characterize the Macroprocessor complex and identify its substrates. In order to study drosha?s function, I have previously employed retroviral siRNA-producing vectors to reduce drosha protein levels in HeLa cells, and found that knockdown cells show reduced viability by a colony formation assay. Rnt1, the sole RNAse III in budding yeast, has several mRNA substrates. To determine if drosha, too, has a role in mRNA processing, I conducted an expression analysis of the knockdowns using microarrays. There were a total of 44 probe sets identified, 17 of which increased in the drosha knockdowns, and 27 of which decreased. One of the 27 targets that decreased (c-jun) was identified with two different probe sets. Another decreasing target in all four comparisons was, as expected, the drosha mRNA itself. None of the targets (except drosha) display homology to any of the siRNA constructs, making it unlikely that they are off-target effects. I am building on my substantial body of preliminary data, capitalizing on my unique and powerful reagents (the affinity-purified anti-drosha antibody and the anti-drosha siRNA knockdown constructs), as well as the extensive biochemical and mass spectrometric resources of the Chait lab to characterize the role of drosha in the Macroprocessor complex. My specific aims include: 1. Isolation and identification of drosha substrates 2. Determination of drosha?s dynamics within the cell cycle