Molecular Basis of mRNA Export

 描述（由申请人提供）：进化的一个重要标志是遗传信息在细胞核中的封闭。细胞核中的复制和转录以及细胞质中的翻译的空间分离强加了在这两个区室之间运输数千个大分子的要求。核孔复合物（NPC）是允许跨核膜双向大分子交换的唯一通道，因此作为遗传信息从DNA到RNA再到蛋白质流动的关键调节剂发挥作用。NPC是一个巨大的运输通道，由约30种不同的蛋白质构成，称为核孔蛋白（nups），它们组装成6个进化上保守的亚复合物。由于8重对称性，每个亚复合物以多个拷贝存在于完全组装的NPC中，使得整个组装体在脊椎动物中达到约120 MDa的非凡分子量。NPC外壳是一种直接与核膜的内外膜相互作用的蛋白质外壳，由多个拷贝的杂七聚体外壳核孔蛋白复合物（CNC）组装而成。NPC外壳锚定具有中央转运通道的甜甜圈形NPC核心，并在其暴露的核质和细胞质表面上为不对称核孔蛋白提供关键结合位点，这反过来又将各种转录和mRNA输出机制募集到NPC。这些相关的机制建立了运输方向性，并允许NPC在基因调控中发挥重要作用。因此，不对称核孔蛋白的遗传修饰与多种人类疾病相关，如肿瘤性疾病或对病毒感染的易感性。在高等真核生物中，不对称的细胞质丝核孔蛋白Nup358，在单细胞真核生物中没有同源物，是有效合成分泌蛋白所必需的，这取决于替代RNA输出（ALREX）途径。ALREX mRNA的特征在于信号序列编码区（SSCR），其编码蛋白质靶向内质网所需的短疏水性共有多肽。在NPC的细胞质面，Nup358通过直接结合其SSCR与ALREX mRNA相互作用，并且是其有效翻译所必需的。在人类Nup358中，介导SSCR结合的结构域已被鉴定为急性坏死性脑病（ANE）遗传形式的易感性位点，ANE是一种罕见但严重的疾病，由一些儿童在常见病毒感染后发展而来。虽然ANE的病因仍然未知，但促炎细胞因子水平的增加与Nup358在ALREX途径中的共同参与以及ANE支持分泌蛋白表达的失调可能起相关作用的假设。在这个建议中，我们试图阐明细胞质面的原子结构， 鼻咽癌的研究，并建立Nup358调控ALREX通路的分子机制，同时评估其与分泌蛋白合成和ANE的相关性。这些目标将通过生物化学、结构和功能方法的组合来实现，这些方法旨在剖析相关核孔蛋白（包括野生型和ANE相关Nup358变体）与SSCR RNA之间的分子相互作用。总的来说，我们的研究将有助于理解基本的mRNA输出机制，特别是与分泌蛋白质组的合成相关，并可能为与mRNA输出功能障碍相关的人类疾病和ANE（一种经常无法治疗且最终致命的核孔蛋白疾病）的发展提供治疗上重要的线索。

 DESCRIPTION (provided by applicant): One of the great hallmarks of evolution is the enclosure of genetic information in the nucleus. The spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that allow bi-directional macromolecular exchange across the nuclear envelope and thus function as key regulators of the flow of genetic information from DNA to RNA to protein. The NPC is a massive transport channel that is constructed by ~30 distinct proteins, termed nucleoporins (nups), which assemble into six evolutionarily conserved subcomplexes. Due to 8-fold symmetry, each subcomplex is present in multiple copies in the fully assembled NPC, such that the entire assembly reaches the extraordinary molecular mass of ~120 MDa in vertebrates. The NPC coat, a protein shell that directly interacts with the inner and outer membranes of the nuclear envelope, is assembled from multiple copies of the hetero-heptameric coat nucleoporin complex (CNC). The NPC coat anchors the donut-shaped NPC core harboring the central transport channel, and provides key binding sites for asymmetric nucleoporins on its exposed nucleoplasmic and cytoplasmic faces that, in turn, recruit various transcription and mRNA export machineries to the NPC. These associated machineries establish transport directionality and allow the NPC to play a major role in gene regulation. As such, it is unsurprising that genetic modifications of asymmetric nucleoporins have been associated with a diverse set of human conditions, such as neoplastic diseases or susceptibility to escalating viral infections. In higher eukaryotes, the asymmetric cytoplasmic filament nucleoporin Nup358, which has no homolog in single-cell eukaryotes, is required for the efficient synthesis of secretory proteins that depends on an alternative RNA export (ALREX) pathway. ALREX mRNAs are characterized by a signal sequence-coding region (SSCR), which encodes the short hydrophobic consensus polypeptide required for protein targeting to the endoplasmic reticulum. At the cytoplasmic face of the NPC, Nup358 interacts with ALREX mRNAs through direct binding to their SSCR and is required for their efficient translation. In human Nup358, the domain mediating SSCR binding has been identified as a susceptibility locus for a hereditary form of acute necrotizing encephalopathy (ANE), a rare but severe condition developed by some children upon common viral infections. Although the etiology of ANE remains unknown, increased levels of pro-inflammatory cytokines in combination with the shared involvement of Nup358 in the ALREX pathway as well as ANE support the hypothesis that a deregulation of secretory protein expression may play a relevant role. In this proposal, we seek to elucidate the atomic architecture of the cytoplasmic face of the NPC and establish the molecular mechanism of ALREX pathway regulation by Nup358 while assessing its relevance to secretory protein synthesis and ANE. These goals will be achieved through a combination of biochemical, structural and functional approaches designed to dissect molecular interactions between the relevant nucleoporins including wild type and ANE-associated Nup358 variants and SSCR RNA. Overall, our research will contribute to the understanding of fundamental mRNA export mechanisms with particular relevance to the synthesis of the secretory proteome and may provide therapeutically important clues to human diseases associated with mRNA export dysfunction and the development of ANE, a frequently untreatable and ultimately fatal nucleoporin disease.