DROSHA FUNCTION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 MicroRNA是一种小的非编码调节RNA，与发育和癌症有关。MicroRNA前体由微处理器处理，微处理器是由内切核酸酶drosha及其伴侣DGCR 8蛋白组成的复合物。Drosha也是另一个更大的核复合体的一部分，以下称为Macroprocessor。Macroprocessor包含多种RNA结合和解旋因子以及与人类肿瘤相关的RNA结合蛋白EWS，但其功能和底物的身份尚不清楚。我打算描述宏处理器复合体的特征并确定其基质。为了研究卓沙？为了研究Drosha的功能，我以前曾使用逆转录病毒siRNA生产载体来降低HeLa细胞中的Drosha蛋白水平，并通过集落形成试验发现敲低的细胞显示出降低的生存力。 Rnt 1是芽殖酵母中唯一的RNA酶III，具有几种mRNA底物。为了确定drosha是否也在mRNA加工中发挥作用，我使用微阵列对敲除进行了表达分析。总共发现了44个探针组，其中17个在Drosha击倒中增加，其中27个减少。用两种不同的探针组鉴定了减少的27个靶标之一（c-jun）。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。没有一个靶标（除了drosha）显示出与任何siRNA构建体的同源性，使得它们不太可能是脱靶效应。 我正在建立我的大量初步数据，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。到目前为止，我已经分离并鉴定了几种drosha底物/相互作用物，并确定了drosha在细胞周期中的动力学的某些方面。

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.