Drosophila Down Syndrome Cell Adhesion Molecule: A paradigm for revealing hidden splicing codes

果蝇唐氏综合症细胞粘附分子：揭示隐藏剪接代码的范例

• 批准号: BB/T003936/1
• 负责人: Matthias Soller
• 金额: $ 65.37万
• 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: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT003936%2F1
• 关键词: Drosophila; Down; Syndrome; Cell; Adhesion

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 organization into protein-coding DNA sequences, termed exons, which are separated by non-coding introns. During splicing, introns are excised from the pre-messenger RNA (mRNA) transcript by the spliceosome and exons are joined to form the mature 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 (AS), is used in most human genes and is an important mechanism to build complex organisms with comparatively few genes. AS is particularly prevalent in the brain and changes during aging. Mis-regulation of AS is also associated with various human diseases, including cancer, metabolic disorders and neurodegeneration.Fidelity of splicing rests critically on accurate reading of 'splicing information' in non-coding regions of the pre-mRNA. Paradoxically, introns are often very large and contain numerous sequence motifs that look like splice sites. Hence, the splicing information is encrypted in a code of short sequence motifs that we do not understand very well. As the splicing process is very complex, it is also vulnerable to cause human disease from mutations present in our genomes that result in aberrant splicing. In fact, about 15% of genetic human disease is caused by mutations in splice sites, but considering all regulatory elements involved in splicing, estimates range up to 50%. However, a drug consisting of a short stretch of nucleotides has recently been approved in the US and the EU for correction of splicing in the Spinal Muscular Atrophy (SMA) gene. Since such drugs can be directed to any part in the genome, many cases of aberrant splicing causing human disease could potentially be corrected. To make full use of this technology we need to understand the splicing code.The fruit fly Drosophila has proven an excellent and cost-effective genetic model for deducing basic biological processes as illustrated by the 2017 Nobel prize award. To discover fundamental splicing codes the Down Syndrome Cell Adhesion Molecule (Dscam) is an excellent model gene, because it is extensively alternatively spliced in four arrays of variable exons where only one exon is chosen for inclusion in the mature mRNA. This way, 36'016 different protein isoforms can be generated, which are more proteins from one gene than genes are present in the genome. This diversity is essential for development of the brain, but also in the immune system for recognition and clearance of pathogens such as bacteria. The most central questions regarding Dscam AS is why exons in the variable clusters are not spliced together despite having consensus splice sites and how one variable exon is chosen. We now have developed a toolkit in the fruit fly Drosophila that allows us to test a) whether splicing together of variable exons is prevented by splicing signals being too close together to allow for assembly of a functional splicesome, b) whether long-range base-pairings are key to Dscam AS to bring a variable exon into the proximity of flanking constant exons and c) whether each variable cluster contains unique regulatory sequences that restrict splicing to specific parts of a gene.From these experiments we will learn about fundamental mechanism involved in AS regulation and how their mis-regulation can lead to human disease. Our results will be instrumental for elucidating the splicing code to instruct how human disease caused by splicing errors can be corrected.

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信使 RNA (mRNA) 转录物中切除，外显子被连接形成成熟的 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“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（AS），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。 AS 在大脑中尤其普遍，并且会随着衰老而发生变化。 AS 的错误调节还与多种人类疾病有关，包括癌症、代谢紊乱和神经变性。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。矛盾的是，内含子通常非常大，并且包含许多看起来像剪接位点的序列基序。因此，剪接信息被加密在我们不太理解的短序列基序代码中。由于剪接过程非常复杂，因此也很容易因基因组中存在的突变导致异常剪接而导致人类疾病。事实上，大约 15% 的人类遗传疾病是由剪接位点突变引起的，但考虑到剪接涉及的所有调控元件，估计范围高达 50%。然而，一种由短核苷酸组成的药物最近在美国和欧盟获得批准，用于纠正脊髓性肌萎缩症 (SMA) 基因的剪接。由于此类药物可以针对基因组中的任何部分，因此许多导致人类疾病的异常剪接病例都有可能得到纠正。为了充分利用这项技术，我们需要了解剪接代码。果蝇已被证明是一种出色且具有成本效益的遗传模型，可用于推导基本生物过程，正如 2017 年诺贝尔奖所证明的那样。为了发现基本的剪接代码，唐氏综合症细胞粘附分子 (Dscam) 是一种出色的模型基因，因为它广泛选择性剪接在四个可变外显子阵列中，其中仅选择一个外显子包含在成熟 mRNA 中。这样，可以生成 36,016 种不同的蛋白质亚型，这些蛋白质来自一个基因，比基因组中存在的基因还要多。这种多样性对于大脑的发育至关重要，而且对于免疫系统识别和清除细菌等病原体也至关重要。关于 Dscam AS 的最核心问题是，尽管具有共有剪接位点，但可变簇中的外显子为何不剪接在一起，以及如何选择一个可变外显子。我们现在在果蝇果蝇中开发了一个工具包，使我们能够测试a）是否通过剪接信号太靠近而无法组装功能性剪接体来阻止可变外显子的剪接在一起，b）是否长程碱基-配对是 Dscam AS 将可变外显子带入侧翼恒定外显子附近的关键，以及 c) 每个可变簇是否包含限制剪接到基因特定部分的独特调控序列。通过实验，我们将了解 AS 调节的基本机制以及它们的错误调节如何导致人类疾病。我们的结果将有助于阐明剪接代码，以指导如何纠正剪接错误引起的人类疾病。