Investigating how alternative splicing processes affect cartilage biology from development to old age

选择性RNA剪接(AS)和选择性聚腺苷酸化(AP)是真核生物基因转录后调控的两种重要机制。在人类中，超过95%的多外显子基因是交替剪接的，允许一个基因编码具有不同(有时是拮抗的)功能、不同的亚细胞定位和不同的蛋白质-蛋白质相互作用的多种蛋白质亚型。交替的多聚腺苷酸化会导致异源转录库，这些转录库共享相似的蛋白质编码区，但具有不同长度的3‘UTRs和转录本的稳定性。这两种调节机制对正常的生理过程至关重要，包括组织发育、分化和动态平衡。转录后基因调控在衰老过程中受到失调，对小鼠组织的全球分析表明，随着年龄的增长，选择性剪接基因的数量增加。选择性剪接被认为是人类衰老过程中的一个重要驱动因素，因为它涉及调控控制衰老的各种标志的基因，包括衰老相关基因。此外，剪接因子表达的改变以及mRNA剪接/多聚腺苷的异常是一些与年龄相关的病理的共同特征，包括癌症、心血管疾病和神经退行性变。有针对性的研究强调AS和AP是软骨发育和分化的重要调节因子。例如，几个软骨细胞外基质基因的剪接受到发育调节，导致组成软骨基质的蛋白质亚型发生变化。然而，选择性剪接和多聚腺苷酸化在软骨发育、健康和年龄相关功能障碍中的作用尚未在全球转录组水平上进行探索。这项研究的目的是在全球范围内描述在人类生命过程中软骨中表达的所有转录异构体。学生将使用综合生物信息学分析和基于实验室的细胞和分子生物学技术相结合：1.全球表征软骨中发生在胚胎(6wpc，17wpc)和出生后(2-18年)以及从中年到老年过渡期间(50-85年)的选择性剪接和多聚腺苷酸化事件。2.确定骨髓间充质干细胞(MSCs)在体外分化为软骨过程中发生的剪接和多聚腺苷的变化。3.检测软骨关键基因的特定年龄相关基因亚型对软骨内稳态和对生理刺激反应的影响。利用现有的软骨RNA测序数据集，生物信息学工具将用于识别软骨的发育、分化以及与年龄相关的选择性剪接和多聚腺苷化异构体。在线工具(如DoChaP和PFAM)将用于预测剪接事件对编码蛋白质的影响。为了从机械上了解剪接在发育和老化过程中的变化，将扫描信使核糖核酸亚型以寻找剪接调节因子的结合位点。聚焦于关键的软骨基因，大约100个选定的与年龄相关的AS和AP事件将在新的老龄化队列中使用毛细管凝胶电泳法进行验证，并通过蛋白质印迹检测对关键蛋白质子集的影响。分别用AONS和CRISPR-Cas9系统在原代软骨细胞、SW1353人软骨肉瘤细胞和MSCs向软骨分化过程中操纵5个AS和5个AP事件，通过qRT-PCR、Western印迹、免疫组织化学和生化分析检测对软骨细胞分化、增殖、凋亡、衰老和对病理刺激的反应的影响。

Alternative RNA splicing (AS) and alternative polyadenylation (AP) are two essential mechanisms for the post-transcriptional control of gene expression in eukaryotes. Over 95% of multi-exonic genes are alternatively spliced in humans, allowing a single gene to encode multiple protein isoforms with different (sometimes antagonistic) functions, distinct subcellular localisations and diverse protein-protein interactions. Alternative polyadenylation results in heterogenous transcript pools that share similar protein coding regions but have different length 3'UTRs and transcript stabilities. These two regulatory mechanisms are crucial for normal physiological processes including tissue development, differentiation and homeostasis. Post-transcriptional gene regulation is dysregulated during ageing, and global analysis of mouse tissues revealed that there are an increased number of alternatively spliced genes with age. Alternative splicing is hypothesised to be an important driver in the ageing process in humans as it is involved in regulating genes that control various hallmarks of ageing, including senescence-associated genes. Furthermore, altered expression of splicing factors, as well as abnormal mRNA splicing/polyadenylation, is a common feature of several age-related pathologies including cancer, cardiovascular disease and neurodegeneration. Targeted studies have highlighted AS and AP as important modulators of cartilage development and differentiation. For example, splicing of several cartilage extracellular matrix genes is developmentally regulated, leading to changes in the protein isoforms that compose the cartilage matrix. However, the role of alternative splicing and polyadenylation in cartilage development, health and age-related dysfunction has not yet been explored at the global transcriptome level. The aim of this studentship is globally characterise all transcript isoforms expressed in cartilage across the human lifecourse. The students will use a combination of comprehensive bioinformatics analysis and laboratory based cell and molecular biology techniques to; 1. Globally characterise alternative splicing and polyadenylation events in cartilage that occur during embryonic (6wpc, 17wpc) and postnatal development (2-18ys), and during the transition from middle to old age (50-85ys). 2. Identify splicing and polyadenylation changes that take place during the in vitro differentiation of mesenchymal stem cells (MSCs) into cartilage. 3. Examine the effect that specific age-associated mRNA isoforms of key cartilage genes have on cartilage homeostasis and response to physiological stimuli. Using existing cartilage RNA sequencing datasets, bioinformatics tools will be used to identify cartilage development, differentiation and age-related alternative splice and polyadenylation isoforms. Online tools (e.g. DoChaP and PFAM) will be used to predict the effect of splicing events on the encoded proteins. To mechanistically understanding how splicing changes during development and ageing, mRNA isoforms will be scanned for the binding sites of splicing regulators. Focusing on key cartilage genes, ~100 selected age-related AS and AP events will be validated using capillary gel electrophoresis in a new ageing cohort, with effects on a subset of key proteins tested by western blot. Five AS and five AP events will be manipulated using AONs and the CRISPR-Cas9 system respectively in primary chondrocytes, the SW1353 human chondrosarcoma cells and during chondrogenic differentiation of MSCs, with the effects on chondrocyte differentiation, proliferation, apoptosis, senescence and response to pathological stimuli examined using qRT-PCR, western blot, immunohistochemistry and biochemical assays.