DROSHA FUNCTION

德罗莎功能

• 批准号: 8361515
• 负责人: ERICA Y JACOBS
• 金额: $ 3.26万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361515
• 关键词: Affinity; Antibodies; Biochemical; Biological Assay; Cell Cycle; Cells; Complex; Data; Development; Functional RNA; Funding; Grant; Hela Cells; Human; JUN gene; Malignant Neoplasms; Messenger RNA; MicroRNAs; Microprocessor; National Center for Research Resources; Nuclear; Principal Investigator; Process; Proteins; RNA Binding; RNA-Binding Protein EWS; RNASE3L gene; Reagent; Research; Research Infrastructure; Resources; Role; Saccharomycetales; Small Interfering RNA; Source; United States National Institutes of Health; cost; endonuclease; macromolecule; tumor; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Thus far I have isolated and identified several drosha substrates/interactors and determined some aspects of drosha's dynamics within the cell cycle.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 MicroRNA 是与发育和癌症有关的小型非编码调节 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 蛋白组成的复合体。 Drosha 也是另一个更大的核复合体的一部分，以下称为宏处理器。宏处理器含有多种 RNA 结合和解旋因子以及 EWS（一种与人类肿瘤相关的 RNA 结合蛋白），但其功能及其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其基底。为了研究 d​​rosha 的功能，我之前使用逆转录病毒 siRNA 生成载体来降低 HeLa 细胞中的 drosha 蛋白水平，并通过集落形成测定发现敲除细胞显示出活力降低。 Rnt1 是芽殖酵母中唯一的 RNAse III，具有多种 mRNA 底物。为了确定 drosha 是否也在 mRNA 加工中发挥作用，我使用微阵列对敲低进行了表达分析。总共鉴定了 44 个探针组，其中 17 个在 drosha 敲低中增加，27 个减少。使用两种不同的探针组鉴定出 27 个下降的靶标之一 (c-jun)。正如预期的那样，所有四次比较中另一个下降的目标是 drosha mRNA 本身。没有一个靶标（drosha 除外）与任何 siRNA 构建体表现出同源性，因此它们不太可能是脱靶效应。 我正在建立大量的初步数据，利用我独特而强大的试剂（亲和纯化的抗drosha抗体和抗drosha siRNA敲低构建体），以及Chait广泛的生化和质谱资源实验室来描述 drosha 在宏处理器复合体中的作用。到目前为止，我已经分离和鉴定了几种 drosha 底物/相互作用物，并确定了 drosha 在细胞周期内动力学的某些方面。