Multimerisation of ELAV/Hu proteins - a key mechanism ensuring fidelity of alternative splicing regulation

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The exciting prospect of exploiting genome information for personalized medicine critically depends on the extent to which we understand the regulatory information residing outside the protein-coding regions of the genome. A unique feature of genes in eukaryotic organisms is their organisation into protein-coding DNA sequences, termed exons, which are separated by non-coding introns. During splicing, introns are excised from the pre-mRNA transcript by the spliceosome and exons are joined to form the mature messenger RNA (mRNA). A functional protein can then be made from the mRNA, but only if splicing controlled by hundreds of proteins has accurately taken place. The unique organization of eukaryotic "genes in pieces" further allows exons to be included in one mRNA from a particular gene, but excluded in another. This process, termed alternative splicing, is used in most human genes and is an important mechanism to build complex organisms with comparatively few genes. Alternative splicing is particularly prevalent in the brain and changes during aging. Misregulation of alternative splicing is also associated with various human diseases, including cancer and neurodegeneration.Fidelity of splicing rests critically on accurate reading of 'splicing information' in non-coding regions of the pre-mRNA. The splicing information is encrypted in a code of short sequence motifs that we do not understand very well. Paradoxically, genes that are spliced differently appear to have similar regulatory sequences. Evidently, evolution has generated a strategy to decrypt such splicing information, but it is upon us now to decipher this code. Knowing the splicing code will allow us to interpret genome sequences of regulatory regions, which are the sequences differing most among individuals.The splicing code is read by RNA binding proteins shaped complementary to short parts on the RNA surface. Imagine a gecko, whose toes tightly attach to the slightly uneven surface of a wall. Only the combinatorial use of all its toes allow it to run up the wall. Accordingly, a concept has emerged implementing combinatorial binding of RNA binding proteins for generating an extended surface to bind to RNA and thereby providing specificity in RNA recognition and alternative splicing regulation. To date, however, little is known how RNA binding proteins assemble to provide this level of specificity.To understand this novel mechanism in alternative splicing regulation, work in our laboratory has focused on ELAV (Embryonic Lethal Abnormal Visual system) proteins originally identified in Drosophila, consisting of a family of highly related proteins with homologues in humans called Hu proteins. ELAV/Hu proteins are prototype RNA binding proteins containing three RNA Recognition Motifs (RRM) and are predominantly expressed in neurons. An inherent property of ELAV/Hu proteins is their ability to multimerize. Hence, ELAV/Hu proteins represent an ideal system to determine the structural framework of how multiple copies of RNA binding proteins adopt a complementary shape to RNA for gene-specifically regulating alternative splicing.We have recently obtained a 3D tetramer structure of ELAV RRM3, the main multimerization domain, allowing now to dissect multimerization and RNA binding functions that reside in different parts of the structure. We therefore propose to a) determine the biochemical and biophysical properties leading to multimerization, b) determine how multimerization contributes to gene-specific alternative splicing regulation using Drosophila transgenes and c) how ELAV connects with core pre-mRNA processing machinery.From these experiments we will learn about fundamental principles involved in alternative splicing regulation and how their misregulation can lead, in the case of ELAV/Hu proteins, to neurological disease. Our results will be instrumental for elucidating the splicing code and its interpretation by RNA binding proteins during aging.