DROSHA FUNCTION

德罗莎功能

• 批准号: 7355137
• 负责人: ERICA Y JACOBS
• 金额: $ 1.23万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355137
• 关键词: DROSHA; FUNCTION

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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。MicroRNAs是与发育和癌症有关的小的非编码调控rna。MicroRNA前体由微处理器处理，微处理器是一种由核酸内切酶drosha及其伙伴DGCR8蛋白组成的复合物。德罗沙也是另一个更大的核综合体的一部分，以下称为Macroprocessor。Macroprocessor含有多种RNA结合和解绕因子以及参与人类肿瘤的RNA结合蛋白EWS，但其功能和底物的身份尚不清楚。我打算描述Macroprocessor复合物并确定其底物。为了学习卓莎？我以前曾使用逆转录病毒sirna产生载体来降低HeLa细胞中的drosha蛋白水平，并通过集落形成试验发现，敲除的细胞表现出降低的生存能力。Rnt1是出芽酵母中唯一的RNAse III，具有几种mRNA底物。为了确定drosha是否也在mRNA加工中起作用，我使用微阵列对基因敲低进行了表达分析。共鉴定出44个探针集，其中17个在drosha敲低中增加，27个减少。27个减少的靶标之一（c-jun）被两种不同的探针组确定。正如预期的那样，在所有四种比较中，另一个减少的目标是drosha mRNA本身。没有一个靶标（除了drosha）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。我正在建立在我的大量初步数据的基础上，利用我独特而强大的试剂（亲和纯化的抗drosha抗体和抗drosha siRNA敲低结构），以及Chait实验室广泛的生化和质谱资源来表征drosha在Macroprocessor复合物中的作用。我的具体目标包括：1。2.黄沙底物的分离鉴定。drosha的测定？细胞周期内的S动力学