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Characterization and function of ELAV post-transcriptionally controlled gene networks in neuronal differentiation

ELAV 转录后控制基因网络在神经元分化中的特征和功能

基本信息

批准号：
BB/F000855/1
负责人：
Matthias Soller
金额：
$ 59.48万
依托单位：
University of Birmingham
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FF000855%2F1
关键词：
Characterization function ELAV post transcriptionally

项目摘要

A characteristic of eukaryotic genes is the interruption of the protein coding sequence by non-coding sequences called introns. During transcription of genes into pre-messengerRNA, introns are spliced out and the protein coding pieces, called exons, are joined into messengerRNA (mRNA) that encodes a continuous sequence of the entire protein when translated in the cytoplasm. During the maturation of the mRNA, some exons are variably included in a process called alternative splicing and generate proteins from the same gene that vary in their sequence. Sequencing of the human genome revealed that alternative splicing is abundant and found in at least 60 % of genes. Alternative splicing is most prevalent in the brain and generates enormous molecular diversity. Due to the complexity of the human brain, alternative splicing has been implicated in providing an essential contribution in wireing neurons during development. Neuronal connections in the human brain are not static and can be remodeled when sensory information is processed. Learning and formation of memories further involve changes in the chemical communication between neurons at their contact sites called synapses and also involve remodeling of synapses. To understand how alternative splicing regulation contributes to neuronal connectivity and remodeling of synaptic contacts we use Drosophila as a genetic model system, allowing us to alter gene function in neurons of a developing and performing organism. In particular, we have focused on studying the function of the ELAV, a Drosophila homologue of human Hu proteins and founding member of a family of RNA binding proteins. Although, the essential gene elav is expressed in all neurons, elav mutants have specific defects in axon guidance of midline crossing neurons, in synaptic growth and in photoreceptor neuron development. Consistent the distinct phenotypes of elav mutants, we have found that ELAV is a gene-specific regulator of alternative splicing. Based on the organization of functionally connected prokaryotic genes into transcription units, so called operons, and co-regulated expression of functionally connected genes in the simple eukaryote yeast, it has been proposed that similar principles apply to the regulation of alternative splicing. To test the hypothesis, that co-regulated alternatively spliced genes are functionally connected and to understand the function of ELAV in neurons we will first determine the targets of ELAV. RNA targets bound to ELAV will be purified, amplified and hybridized to Drosophila tiling microarrays, representing the entire Drosophila genome on a single slide as millions of spots of unique sequences. The second step is then to establish functional connections by grouping ELAV target genes according to co-expression at the same developmental stage, annotated gene functions, regulation by the same mechanism and sharing targets with other ELAV family members. In the third step, functions will then be assigned to sets of co-regulated genes through phenotypes of mutants that specifically lack those protein isoforms that are made in the presence of ELAV. The analysis of ELAV regulated genes at genome wide dimensions will yield fundamental insights into functional connections among genes that are involved in brain functions essential to provide quality to life such as learning and memory.

真核生物基因的一个特征是蛋白质编码序列被称为内含子的非编码序列打断。在基因转录成pre-messengerRNA的过程中，内含子被剪接出来，蛋白质编码片段（称为外显子）被连接到messengerRNA （mRNA）中，当在细胞质中翻译时，mRNA编码整个蛋白质的连续序列。在mRNA的成熟过程中，一些外显子被可变地包括在一个称为选择性剪接的过程中，并从相同的基因中产生序列不同的蛋白质。人类基因组测序显示，选择性剪接是丰富的，在至少60%的基因中发现。选择性剪接在大脑中最为普遍，并产生了巨大的分子多样性。由于人类大脑的复杂性，选择性剪接在发育过程中为神经元的连接提供了重要的贡献。人类大脑中的神经元连接不是静态的，在处理感觉信息时可以重塑。学习和记忆的形成进一步涉及神经元之间的化学交流的变化，这些交流是在它们的接触点突触上进行的，也涉及突触的重塑。为了了解选择性剪接调节如何促进神经元连接和突触接触重塑，我们使用果蝇作为遗传模型系统，允许我们改变发育和活动生物体神经元中的基因功能。特别是，我们专注于研究ELAV的功能，ELAV是人类Hu蛋白的果蝇同源物，也是RNA结合蛋白家族的创始成员。尽管必需基因elav在所有神经元中都有表达，但elav突变体在中线交叉神经元的轴突引导、突触生长和光感受器神经元发育中存在特异性缺陷。与elav突变体的不同表型一致，我们发现elav是选择性剪接的基因特异性调节因子。基于将功能连接的原核基因组织成转录单位，即所谓的操纵子，以及在简单真核生物酵母中共同调节功能连接基因的表达，已经提出类似的原理适用于调节选择性剪接。为了验证这一假设，即共调节的选择性剪接基因在功能上相互连接，并了解ELAV在神经元中的功能，我们将首先确定ELAV的靶点。与ELAV结合的RNA靶标将被纯化、扩增并杂交到果蝇平铺微阵列中，在一张载玻片上代表整个果蝇基因组，作为数百万个独特序列的点。第二步是根据在同一发育阶段共表达、注释的基因功能、受相同机制调控以及与其他ELAV家族成员共享靶标对ELAV靶基因进行分组，建立功能联系。在第三步中，通过突变体的表型，功能将被分配给一组共调节基因，这些突变体特异性地缺乏在ELAV存在下产生的蛋白质同种异构体。在基因组范围内对ELAV调节基因的分析将对涉及大脑功能的基因之间的功能联系产生根本性的见解，这些功能对提供学习和记忆等生活质量至关重要。