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

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编码蛋白质的DNA序列，称为外显子，外显子被非编码内含子分开。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，外显子连接形成成熟的mRNA。然后可以从mRNA中合成功能蛋白质，但前提是由数百种蛋白质精确控制的剪接已经发生。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种mRNA中，但排除在另一种mRNA中。这个过程被称为选择性剪接（AS），用于大多数人类基因，是构建基因相对较少的复杂生物体的重要机制。AS在大脑中特别普遍，并在衰老过程中发生变化。AS的错误调节也与多种人类疾病有关，包括癌症、代谢紊乱和神经退行性疾病。剪接的保真度关键在于对前体mRNA非编码区“剪接信息”的准确阅读。奇怪的是，内含子通常非常大，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了解的短序列基序的代码中。由于剪接过程非常复杂，它也容易因我们基因组中存在的导致异常剪接的突变而引起人类疾病。事实上，约15%的人类遗传性疾病是由剪接位点突变引起的，但考虑到所有参与剪接的调控元件，估计高达50%。然而，一种由一小段核苷酸组成的药物最近在美国和欧盟被批准用于纠正脊髓性肌萎缩症（SMA）基因的剪接。由于这类药物可以针对基因组中的任何部分，许多导致人类疾病的异常剪接病例可能会得到纠正。为了充分利用这项技术，我们需要了解剪接密码。果蝇果蝇已经证明了一个优秀的和具有成本效益的遗传模型，用于推导基本的生物过程，如2017年诺贝尔奖所示。为了发现基本剪接密码，唐氏综合征细胞粘附分子（Dscam）是一个很好的模型基因，因为它在四个可变外显子阵列中广泛地选择性剪接，其中仅选择一个外显子包含在成熟mRNA中。这样，可以产生36'016种不同的蛋白质同种型，其是来自一个基因的蛋白质多于基因组中存在的基因。这种多样性对大脑的发育至关重要，但也是免疫系统识别和清除病原体如细菌的关键。关于Dscam AS最核心的问题是为什么可变簇中的外显子不拼接在一起，尽管有共识剪接位点，以及如何选择一个可变外显子。我们现在已经在果蝇中开发了一个工具包，使我们能够测试a）可变外显子的剪接是否被剪接信号太近而不能组装功能性剪接体所阻止，B）长距离碱基配对是否是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）通过这些实验，我们将了解AS调控的基本机制，以及它们的错误调控如何导致人类疾病。我们的研究结果将有助于阐明剪接密码，指导如何剪接错误引起的人类疾病可以得到纠正。

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.