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中断。在基因转录成前信使RNA的过程中，内含子被剪接，蛋白质编码片段（称为外显子）被连接成信使RNA（mRNA），当在细胞质中翻译时，信使RNA编码整个蛋白质的连续序列。在 mRNA 的成熟过程中，一些外显子不同程度地包含在称为选择性剪接的过程中，并从同一基因生成序列不同的蛋白质。人类基因组测序表明，选择性剪接非常丰富，至少在 60% 的基因中存在。选择性剪接在大脑中最为普遍，并产生巨大的分子多样性。由于人脑的复杂性，选择性剪接在神经元发育过程中的连接中发挥着重要作用。人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。记忆的学习和形成还涉及神经元之间称为突触的接触部位化学通讯的变化，还涉及突触的重塑。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，使我们能够改变发育和执行生物体的神经元中的基因功能。特别是，我们重点研究 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、突触生长和感光神经元发育方面存在特定缺陷。与 elav 突变体的独特表型一致，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，以及在简单真核酵母中共同调节功能连接的基因的表达，有人提出类似的原理适用于选择性剪接的调节。为了检验共同调节的选择性剪接基因在功能上相连的假设，并为了了解 ELAV 在神经元中的功能，我们将首先确定 ELAV 的靶标。与 ELAV 结合的 RNA 靶点将被纯化、扩增并与果蝇平铺微阵列杂交，在单张玻片上将整个果蝇基因组表示为数百万个独特序列点。第二步是根据同一发育阶段的共表达对 ELAV 靶基因进行分组，注释基因功能，通过相同机制进行调节并与其他 ELAV 家族成员共享靶标，从而建立功能连接。在第三步中，通过突变体的表型，将功能分配给一组共同调控的基因，这些突变体特别缺乏在 ELAV 存在下产生的蛋白质亚型。在全基因组范围内对 ELAV 调节基因进行分析，将对基因之间的功能联系产生基本的了解，这些基因涉及大脑功能，这些功能对于提供学习和记忆等生活质量至关重要。