Assessing the impact of ALS Mutations on mRNA Translation in hiPSC-derived Neurons and Neuromuscular Models

肌萎缩性侧索硬化症（ALS）是一种神经退行性疾病，导致上下运动神经元逐渐退化（在Masrori和Van Damme， 2020年进行了综述）。在分子上，它的部分特征是与蛋白质-RNA复合物的破坏相关的渐进式RNA解除管制，最终形成聚集体。这种解除管制可以由几种rna结合蛋白（rbp）的突变引发，如TDP-43 （Chia等人，2018；Taylor等人，2016）和FUS （Deng等人，2014）。许多rbp在与RNA结合时形成生物分子凝聚体，这可以选择性地促进RNA调控（Hallegger et al, 2021）。许多引起als的rbp突变已被证明会改变其凝结倾向，从而增加转变为潜在毒性聚集形式的可能性（Cestra等人，2017年，Taylor等人，2016年；Wiedner和Giudice， 2021年进行了综述）。Ule实验室之前的工作主要集中在TDP-43缩聚的生理作用上，表明它允许特定类型的RNA结合，特别是以高多价方式在长3'UTR区域上组装的能力（Halleger等人，2021）。这种“结合区凝聚物”的形成被证明可以引导TDP-43调节3'端mRNA聚腺苷化的能力。als相关突变微妙地破坏了这种浓缩倾向，研究表明，这也微妙地影响了3'UTR加工，但尚未研究对mRNA翻译的影响。考虑到3' utr在mRNA翻译中的重要性，了解它是如何受到干扰核糖核蛋白（RNP）缩合的影响，以及这种直接的RNA失调是如何促进早期疾病阶段的，将是至关重要的。此外，Ule实验室最近对核糖体分析数据进行了分析，发现ALS患者使用了可选开放阅读框（orf）。替代orf可以增加或减少转录本中典型蛋白的合成，改变其长度，或完全产生新蛋白，因此作为翻译调控的一个重要但经常被忽视的层（详见Orr等人，2020）。了解肌萎缩性侧索硬化症RBP功能障碍对翻译的影响，有助于开发新的治疗方法，在早期阶段阻止疾病进展。我们将研究als引起的rbp突变如何直接影响人类神经肌肉系统细胞模型中的mRNA翻译。该项目的第一个目标将有助于Lieberam和Ule实验室建立的具有TDP-43和mat3突变的人类ipsc衍生神经元模型的表型特征，并使用核糖核酸序列评估其转录组全翻译状态。然后，我们将在相同的模型上使用单个核苷酸分辨率的UV交联和免疫沉淀（iCLIP），并进行生物信息学分析，以确定翻译失调的机制，特别是了解突变体TDP-43和mat3如何改变它们与翻译失调rna的相互作用。我们将使用来自代表性rna的调控元件来开发新的双色荧光报告，以实时成像跟踪翻译放松管制。这些将使我们能够在神经肌肉回路的共同培养模型中研究翻译放松管制。

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease which results in the gradual deterioration of upper and lower motor neurons (reviewed in Masrori and Van Damme, 2020). Molecularly, it is characterised partly by progressive RNA deregulation related to disrupted protein-RNA complexes that culminate in the formation of aggregates. Such deregulation can be initiated by mutations in several RNA-binding proteins (RBPs), such as TDP-43 (Chia et al., 2018; Taylor et al., 2016) and FUS (reviewed in Deng et al., 2014). Many of these RBPs form biomolecular condensates when bound to RNAs, which can selectively contribute to RNA regulation (Hallegger et al, 2021). Many ALS-causing mutations in RBPs have been shown to change their condensation propensity, which can increase the probability of transitioning into potentially toxic aggregated forms (reviewed in Cestra et al., 2017, Taylor et al., 2016; Wiedner and Giudice, 2021). Previous work done by the Ule lab has focused on the physiological roles of TDP-43 condensation, showing that it allows specific types of RNA binding, especially the capacity to assemble on long 3'UTR regions in a highly multivalent manner (Halleger et al., 2021). Formation of such "binding-region condensates" was shown to steer the capacity of TDP-43 to regulate 3' end mRNA polyadenylation. ALS-linked mutations subtly disrupt this condensation propensity and it was shown that this also subtly impacts 3'UTR processing, but the impact on mRNA translation has not been examined. Given the importance of 3'UTRs in mRNA translation, it will be crucial to understand how it is impacted by perturbed ribonucleoprotein (RNP) condensation, and how such direct RNA deregulation contributes to the early disease stages. Additionally, a recent analysis of ribosome profiling data from the Ule lab detected the usage of alternative open reading frames (ORFs) in ALS. Alternative ORFs can increase or decrease the synthesis of the canonical protein from a transcript, alter its length, or create a new protein altogether, serving therefore as an important yet often overlooked layer of translational regulation (reviewed in Orr et al., 2020). Understanding the impact of RBP dysfunction in ALS on translation on translation could enable the development of new therapeutic approaches to stop disease progression in its early stages.We will investigate how ALS-causing mutations in RBPs directly affect mRNA translation in a cellular model of the human neuromuscular system. The first aim of the project will be to contribute to the phenotypic characterisation of human iPSC-derived neuron models with mutations in TDP-43 and Matr3 established by the Lieberam and Ule labs and to assess their transcriptome wide translational status with Ribo-seq. We will then use individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) on the same models and run bioinformatic analyses to identify mechanisms of translational deregulation, specifically to understand how mutant TDP-43 and Matr3 change their interactions with translationally deregulated RNAs. We will use the regulatory elements from representative RNAs to develop new bichromatic fluorescent reporters to follow translational deregulation with live imaging. These will allow us to investigate translational deregulation in a co-culture model for neuromuscular circuits.