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

ELAV/Hu 蛋白的多聚化——确保选择性剪接调控保真度的关键机制

• 批准号: BB/K006827/1
• 负责人: Matthias Soller
• 金额: $ 44.69万
• 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: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK006827%2F1
• 关键词: Multimerisation; ELAV; Hu; proteins; key

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.

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域之外的监管信息的理解程度。真核生物基因的一个独特特征是它们组织成蛋白质编码 DNA 序列，称为外显子，由非编码内含子分隔。在剪接过程中，内含子被剪接体从前 mRNA 转录物中切除，外显子连接形成成熟的信使 RNA (mRNA)。然后可以从 mRNA 中制造出功能性蛋白质，但前提是由数百个蛋白质控制的剪接已准确发生。真核生物“基因片段”的独特组织进一步允许外显子包含在来自特定基因的一个mRNA中，但排除在另一个mRNA中。这个过程被称为选择性剪接，用于大多数人类基因，是用相对较少的基因构建复杂生物体的重要机制。选择性剪接在大脑中尤其普遍，并且会随着衰老而发生变化。选择性剪接的错误调节还与多种人类疾病有关，包括癌症和神经退行性疾病。剪接的保真度关键取决于对前 mRNA 非编码区“剪接信息”的准确读取。剪接信息被加密在我们不太理解的短序列基序代码中。矛盾的是，不同剪接的基因似乎具有相似的调控序列。显然，进化已经产生了一种解密这种剪接信息的策略，但现在我们需要破译这个密码。了解剪接代码将使我们能够解释调控区域的基因组序列，这些序列在个体之间差异最大。剪接代码由形状与 RNA 表面短部分互补的 RNA 结合蛋白读取。想象一只壁虎，它的脚趾紧紧地贴在稍微不平坦的墙壁表面上。只有组合使用所有脚趾才能让它爬上墙壁。因此，出现了实现RNA结合蛋白的组合结合的概念，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，迄今为止，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。为了了解选择性剪接调节的这种新机制，我们实验室的工作重点是最初在果蝇中发现的 ELAV（胚胎致死异常视觉系统）蛋白，该蛋白由一系列高度相关的蛋白组成，与人类的同源物称为 Hu 蛋白。 ELAV/Hu 蛋白是原型 RNA 结合蛋白，含有三个 RNA 识别基序 (RRM)，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，用于确定多个拷贝的 RNA 结合蛋白如何采用与 RNA 互补的形状以进行基因特异性调节可变剪接的结构框架。我们最近获得了 ELAV RRM3（主要多聚化结构域）的 3D 四聚体结构，现在可以剖析位于该结构不同部分的多聚化和 RNA 结合功能。因此，我们建议 a) 确定导致多聚化的生化和生物物理特性，b) 确定多聚化如何利用果蝇转基因促进基因特异性选择性剪接调节，以及 c) ELAV 如何与核心前 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所涉及的基本原理以及它们的错误调节如何导致，在 ELAV/Hu 蛋白，可导致神经系统疾病。我们的结果将有助于阐明衰老过程中 RNA 结合蛋白的剪接代码及其解释。

项目成果

A novel protein domain in an ancestral splicing factor drove the evolution of neural microexons.

祖先剪接因子中的一个新蛋白质结构域驱动了神经微外显子的进化。

• DOI: 10.1038/s41559-019-0813-6
• 发表时间: 2019
• 期刊: Nature ecology & evolution
• 影响因子: 16.8
• 作者: Torres-Méndez A

Thiamethoxam exposure deregulates short ORF gene expression in the honey bee and compromises immune response to bacteria.

• DOI: 10.1038/s41598-020-80620-7
• 发表时间: 2021-01-15
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Decio P;Ustaoglu P;Derecka K;Hardy ICW;Roat TC;Malaspina O;Mongan N;Stöger R;Soller M
• 通讯作者: Soller M

Dynamically expressed single ELAV/Hu orthologue elavl2 of bees is required for learning and memory.

• DOI: 10.1038/s42003-021-02763-1
• 发表时间: 2021-10-28
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Ustaoglu P;Gill JK;Doubovetzky N;Haussmann IU;Dix TC;Arnold R;Devaud JM;Soller M
• 通讯作者: Soller M

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals.

• DOI: 10.1126/sciadv.abk0445
• 发表时间: 2022-01-28
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Torres-Méndez A;Pop S;Bonnal S;Almudi I;Avola A;Roberts RJV;Paolantoni C;Alcaina-Caro A;Martín-Anduaga A;Haussmann IU;Morin V;Casares F;Soller M;Kadener S;Roignant JY;Prieto-Godino L;Irimia M
• 通讯作者: Irimia M

Dynamically expressed ELAV is required for learning and memory in bees

动态表达的 ELAV 是蜜蜂学习和记忆所必需的

• DOI: 10.1101/2021.06.24.449637
• 发表时间: 2021
• 作者: Ustaoglu P