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

项目摘要 该提案的目标是确定核孔复合物（NPC）蛋白Megator的作用 通过确定Mtor影响表观遗传的机制， 果蝇的剂量补偿现象。MTOR构成了 NPC，并似乎形成一个核内基质样结构，在有丝分裂后， 唾液腺核多带化。由于Mtor与RNA生物合成过程的调节有关， 酵母和在剂量补偿的过程中，在果蝇，我检查了Mtor对定位的影响， 一种非编码RNA，是剂量补偿（MSL）复合物roX1的一部分。果蝇MSL 复杂的功能，以上调表达的单一男性X染色体的两倍。使用单分子 在幼虫唾液腺细胞核的RNA FISH中，我检测到核可溶性roX1的增加， 除了在Mtor耗尽条件下的正常roX1靶向之外，还具有X染色体。增加的 发现核可溶性roX1的存在是由于雄性中roX1的转录水平增加 原子核。除了rox1，许多其他X连锁基因表现出男性特异性的表达增加 当Mtor耗竭时，表明正常情况下Mtor的功能是限制剂量补偿基因， 表情目的1将检验Mtor限制男性X染色体上基因表达的假设。 RNA-Seq将在来自对照和Mtor耗尽条件的幼虫唾液腺上进行，以及 来自男性和女性。除了检查基因表达的变化，我还可以检查 通过RNA-Seq和RNA FISH检测RNA加工差异，以便开始研究 Mtor对基因表达的影响。目的2将检验Mtor对基因表达的影响是 依赖于与MSL复合物和核外泌体的组分的相互作用， 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， 在剂量补偿的情况下形成核孔蛋白的核支架。这些目标将有助于阐明 Mtor在发育中的生物体中RNA生物合成中的作用，以及扩大我们的理解 NPC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.