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

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.

Alternative pre-mRNA processing is a major mechanism in generating molecular diversity. In humans, 60-80 % of genes are alternatively spliced and this type of gene regulation is particularly widespread in neurons. The functional consequences of this prominent aspect in regulation of gene expression in neurons, however, are largely unknown. To gain insights into how alternative splicing regulation provides essential functions to neurons, we have focused on the neuronal alternative splicing regulator ELAV, a Drosophila homologue of human Hu proteins. Pioneering work in yeast has revealed that expression of functionally connected genes is coordinately regulated. It has therefore been proposed, that similar principles apply to the regulation of gene expression at the post-transcriptional level. In support of the proposed hypothesis, we have found that the currently known four target genes of gene-specific ELAV (ewg, nrg, arm and elav), that all have homologues in humans, are involved in regulating synaptic growth. Our aim is to characterize ELAV post-transcriptionally controlled gene networks and their function in establishing neuronal connectivity and in presynaptically regulating structural plasticity. These connections will be established by determining the complement of ELAV target genes, their regulation by ELAV and sharing targets with other ELAV family members in differentiating neurons. An integral part in understanding the organization of ELAV regulated gene networks is then to establish functional connections among co-regulated genes based on isoform-specific mutants. The proposed project is at the leading edge of functional genomic approaches aiming to understand post-transcriptionally controlled gene networks at a genomic scale and will significantly enhance our understanding how alternative splicing contributes to neuronal differentiation and function.