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

利用基因组信息进行个性化医疗的令人兴奋的前景，关键取决于我们对基因组蛋白质编码区之外的调控信息的理解程度。真核生物中基因的一个独特特征是它们被组织成蛋白质编码DNA序列，称为外显子，它们被非编码内含子分开。剪接过程中，内含子被剪接体从pre-mRNA转录物中切除，外显子连接形成成熟的信使RNA （mRNA）。一种功能性的蛋白质可以从mRNA中合成，但前提是由数百种蛋白质控制的剪接已经准确地发生了。真核生物“基因片段”的独特组织进一步允许外显子包含在一个特定基因的mRNA中，但排除在另一个基因中。这一过程被称为选择性剪接，在大多数人类基因中使用，是构建具有相对较少基因的复杂生物体的重要机制。选择性剪接在大脑中特别普遍，并在衰老过程中发生变化。选择性剪接的错误调节也与各种人类疾病有关，包括癌症和神经变性。剪接的保真度关键取决于对pre-mRNA非编码区“剪接信息”的准确读取。拼接信息被加密在一个短序列基序的代码中，我们不太了解。矛盾的是，拼接方式不同的基因似乎有着相似的调控序列。显然，进化已经产生了一种解密这种拼接信息的策略，但现在是我们来破译这个密码的时候了。了解剪接密码将使我们能够解释调控区域的基因组序列，这是个体之间差异最大的序列。剪接代码由RNA结合蛋白读取，这些蛋白的形状与RNA表面的短部分互补。想象一只壁虎，它的脚趾紧紧地贴在略微凹凸不平的墙壁表面上。只有组合使用所有的脚趾才能让它爬上墙。因此，出现了一种概念，实现RNA结合蛋白的组合结合，以产生与RNA结合的扩展表面，从而提供RNA识别和选择性剪接调节的特异性。然而，到目前为止，人们对RNA结合蛋白如何组装以提供这种水平的特异性知之甚少。为了理解这种选择性剪接调节的新机制，我们实验室的工作集中在ELAV（胚胎致命异常视觉系统）蛋白上，该蛋白最初在果蝇中发现，由一个与人类同源物高度相关的蛋白家族组成，称为Hu蛋白。ELAV/Hu蛋白是包含三个RNA识别基序（RRM）的原型RNA结合蛋白，主要在神经元中表达。ELAV/Hu蛋白的一个固有特性是它们的多聚化能力。因此，ELAV/Hu蛋白代表了一个理想的系统，可以确定RNA结合蛋白的多拷贝如何采用RNA的互补形状来特异性调节选择性剪接。我们最近获得了ELAV RRM3的三维四聚体结构，这是主要的多聚结构域，现在允许解剖位于结构不同部分的多聚和RNA结合功能。因此，我们提出a)确定导致多聚的生化和生物物理特性，b)确定多聚如何有助于使用果蝇转基因进行基因特异性选择性剪接调节，以及c) ELAV如何与核心pre-mRNA加工机制连接。从这些实验中，我们将了解选择性剪接调节的基本原理，以及它们的错误调节如何导致ELAV/Hu蛋白的神经系统疾病。我们的结果将有助于阐明剪接代码及其在衰老过程中RNA结合蛋白的解释。

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.