The role of nuclear pore component Megator in gene expression and dosage compensation

项目摘要 这项提议的目标是确定核孔复合体(NPC)蛋白Megator的作用 (MTOR)通过确定mTOR影响表观遗传学的机制来调节基因表达 果蝇的剂量补偿现象。MTOR构成了核子篮子 在有丝分裂后，它似乎形成一种核内基质样结构，沿着染色质结合， 唾液腺核多角化。由于mTOR已被牵连到RNA生物发生过程的调节中 在果蝇的剂量补偿过程中，我检测了mTOR对其定位的影响。 一种非编码的RNA，是剂量补偿(MSL)复合体roX1的一部分。果蝇Msl 复合体的功能是将单个男性X染色体的表达上调两倍。使用单分子 在鱼类幼虫唾液腺细胞核中，我检测到核中可溶性roX1的增加，这与 在mTOR耗尽的条件下，除了正常的roX1靶向外，还具有X染色体。增加的 核可溶性roX1的存在是由于男性roX1转录水平增加所致。 原子核。除rox1外，许多其他与X相关的基因表现出男性特有的表达增加 当mTOR耗尽时，提示正常的mTOR功能限制了剂量补偿基因 表情。目的1验证mTOR限制男性X染色体基因表达的假设。 RNA-Seq将在对照和mTOR耗尽条件下的幼虫唾液腺以及 无论是雄性还是雌性。除了检查基因表达的变化，我还将能够检查 RNA-Seq和RNA FISH对RNA加工的差异，以开始对其机制的研究 MTOR对基因表达的影响。目标2将检验这样一个假设，即mTOR对基因表达的影响是 依赖于与MSL复合体和核外切体成分的相互作用，使用分析 用于染色质结合和遗传相互作用。这些研究将揭示一种新的基因调控作用 在剂量补偿的背景下形成核孔素的核支架。这些目的将有助于阐明 MTOR在发育中生物体的RNA生物发生中的作用以及扩大我们的理解 全国人大在基因调控中的作用。

Project Summary The goal of this proposal is to determine the role of the nuclear pore complex (NPC) protein Megator (Mtor) in regulation of gene expression by determining the mechanism via which Mtor influences the epigenetic phenomenon of dosage compensation in Drosophila melanogaster. Mtor makes up the nuclear basket of the NPC and appears to form an intranuclear matrix-like structure that, binds along chromatin in post-mitotic, polytenized salivary gland nuclei. Since Mtor has been implicated in regulation of RNA biogenesis processes in yeast and in the process of dosage compensation in Drosophila, I examined the effect of Mtor on localization of a non-coding RNA that is part of the dosage compensation (MSL) complex, roX1. The Drosophila MSL complex functions to upregulate expression of the single male X chromosome two-fold. Using single molecule RNA FISH in larval salivary gland nuclei, I detected an increase in nuclear soluble roX1 that is not associated with the X chromosome in addition to normal roX1 targeting in Mtor-depleted conditions. The increased presence of nuclear soluble roX1 was found to be due to increased levels of transcription of roX1 in male nuclei. In addition to rox1, a number of other X-linked genes exhibited a male-specific increase in expression upon depletion of Mtor, suggesting that normally Mtor functions to restrict dosage compensated gene expression. Aim 1 will test the hypothesis that Mtor restricts gene expression on the male X chromosome. RNA- Seq will be performed on larval salivary glands from both control and Mtor depleted conditions as well as from both males and females. In addition to examining gene expression changes, I will also be able to examine RNA processing differences by RNA-Seq and RNA FISH in order to begin investigation into the mechanism of Mtor’s effects on gene expression. Aim 2 will test the hypothesis that Mtor’s effects on gene expression are dependent on an interaction with components of the MSL complex and of the nuclear exosome, using assays for chromatin binding and for genetic interactions. These studies will uncover a novel gene regulatory role for a nuclear-scaffold forming nucleoporin in the context of dosage compensation. These aims will help to elucidate a role for Mtor in RNA biogenesis in the context of a developing organism as well as expand our understanding of the NPC’s role in gene regulation.