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.

真核基因的特征是通过称为内含子的非编码序列的蛋白质编码序列中断。在将基因转录到前元素中时，内含子被剪接，并将蛋白质编码片（称为外显子）连接到墨西哥（mRNA）中，该蛋白质（mRNA）在细胞质中翻译时编码整个蛋白质的连续序列。在mRNA的成熟过程中，一些外显子在称为替代剪接的过程中多样包含，并从同一基因中产生蛋白质的序列变化。人类基因组的测序表明，替代剪接很丰富，并且在至少60％的基因中发现。替代剪接在大脑中最普遍，并产生巨大的分子多样性。由于人脑的复杂性，替代剪接涉及在发育过程中为电线神经元提供基本贡献。人脑中的神经元连接不是静态的，当处理感觉信息时可以重塑。记忆的学习和形成进一步涉及神经元之间的化学通信在其接触部位称为突触的变化，还涉及突触的重塑。为了了解替代剪接调节如何有助于神经元连通性和突触接触的重塑，我们使用果蝇作为遗传模型系统，从而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，我们专注于研究人HU蛋白的果蝇同源物和RNA结合蛋白家族的创始成员Elav的功能。尽管在所有神经元中表达了必需基因ELAV，但Elav突变体在中线跨线神经元，突触生长和感光受体神经元发育中具有特定缺陷。一致的Elav突变体的不同表型，我们发现Elav是替代剪接的基因特异性调节剂。基于功能连接的原核基因的组织为转录单元，所谓的操纵子，以及在简单的真核生物酵母中的功能连接基因的共同调节表达，已经提出，类似的原理适用于调节替代剪接。为了检验该假设，共同调节的剪接基因在功能上连接并了解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靶标将被纯化，扩增和杂交到果蝇瓷砖微阵列，以数百万个独特的序列的位置代表单个幻灯片上的整个果蝇基因组。然后，第二步是通过根据同一发育阶段的共表达，注释的基因功能，相同机制的调节并与其他Elav家族成员共享目标来建立功能连接。在第三步中，将通过突变体的表型将函数分配给共同调节的基因集，这些突变体特别缺乏在ELAV存在下制造的蛋白质同工型。对基因组宽维的ELAV调节基因的分析将产生对与学习和记忆等质量至关重要的脑功能相关基因之间功能联系的基本见解。