Using microRNA-target chimeras to study post-transcriptional gene regulation in the mammalian CNS

使用 microRNA 靶标嵌合体研究哺乳动物中枢神经系统的转录后基因调控

• 批准号: 10154004
• 负责人: William Thomas Mills
• 金额: $ 4.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-21 至 2023-12-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10154004
• 关键词: 8 year old; Affect; Area; Behavior; Binding Sites; Biogenesis; Bioinformatics; Biological Process; Brain; Brain-Derived Neurotrophic Factor; Child; Chimera organism; Cognition Disorders; Complement; Complex; Cre driver; Defect; Dendritic Spines; Detection; Development; Developmental Disabilities; Disease; Engineering; FMR1; Family; Fragile X Syndrome; Gene Silencing; Gene Targeting; Genetic; Genetic Transcription; Growth; Heterogeneity; High-Throughput Nucleotide Sequencing; Immunoprecipitation; Impaired cognition; In Vitro; Knockout Mice; Learning; Ligation; Light; Malignant Neoplasms; Mediating; Mediator of activation protein; Memory; Memory impairment; Messenger RNA; Metabolism; Methods; MicroRNAs; Modeling; Molecular; Morphogenesis; Morphology; Mus; Nature; Nervous system structure; Neuronal Plasticity; Neurons; Neurosciences; Nucleotides; Pathway interactions; Physiological; Post-Transcriptional Regulation; Process; Prosencephalon; Protein Biosynthesis; Protein Deficiency; Proteins; Protocols documentation; RNA; RNA Binding; RNA-Binding Proteins; Repression; Research; Resistance; Resolution; Role; Shapes; Signal Transduction; Site; Specificity; Synapses; Synaptic Transmission; Testing; Time; Transcript; Translating; Translations; United States; Work; autism spectrum disorder; base; behavioral phenotyping; brain cell; cell type; cognitive function; crosslink; excitatory neuron; genome-wide; improved; in vivo; insight; knock-down; lens; long term memory; mouse model; overexpression; preference; transcriptome; transcriptome sequencing

Amongst the most intriguing and complex questions in neuroscience is that concerning the molecular mechanisms by which synapses participate in learning and memory. Beginning as early as the 1960s, a growing body of evidence indicates that new protein synthesis is required for learning and long-term memory formation.1 Additionally, it has been shown that activity-dependent protein synthesis is highly specific, as only a small subset of available transcripts are translated following neuronal activity.2,3 There is also evidence that local protein synthesis at synapses mediates plasticity and that perturbations in translation are associated with disorders of cognitive function such as autism.4,5 The realization of the proximity of protein synthesis to the sites of synaptic transmission, the specificity of transcripts that are translated, and its disruption in disease has underscored the importance of understanding the nature of post-transcriptional regulatory mechanisms that shape the complement of proteins in neurons and at synapses. Since their discovery in 1993, microRNAs (miRNAs) have been appreciated for their breadth of function as post-transcriptional regulators of protein synthesis by translation inhibition and transcript destabilization.6 miRNAs regulate neuronal plasticity and dendritic spine morphogenesis, are implicated in higher-order brain functions such as memory and cognitive dysfunction, and proteins involved in miRNA biogenesis and function are found in neurons, including near synapses.1 The evolutionarily conserved let-7 family of miRNAs has emerged as a critical mediator of post-transcriptional gene regulation in many growth-related processes including developmental timing (C. elegans7 and D. melanogaster8,9), body axis programming (M. musculus10), metabolism (M. musculus11), and cancer (M. musculus12 and H. sapiens13). The let-7 family of miRNAs are highly abundant in mature differentiated neurons and work from our lab and others has shown that let-7 miRNA levels can be regulated by neuronal activity3,14-16 and are disrupted in a mouse model lacking the fragile X mental retardation protein (FMRP).31 However, an approach to unambiguously determine the genome-wide identity of mRNA targets for let-7 and other miRNAs has not been possible until recently. This project employs a modified version of the recently developed CLEAR-CLIP technique17 that involves cross-linking and immunoprecipitation followed by intermolecular ligation of endogenous RNAs bound to Argonaute and high-throughput sequencing (CIMERA- seq). CIMERA-seq will allow for critical miRNA targets and miRNA-regulatory mechanisms governing protein synthesis in the mammalian CNS to be explored in great detail. The hypothesis of this proposal is that lowered let-7 miRNA levels observed in vitro and in vivo in the FMRP-deficient brain will produce changes in the miRNA- target profile consistent with altered neuronal plasticity, synapse overgrowth, and protein synthesis observed in a mouse model lacking FMRP.

神经科学中最有趣和最复杂的问题之一是关于分子的， 突触参与学习和记忆的机制。早在20世纪60年代， 大量证据表明，新的蛋白质合成是学习和长期记忆形成所必需的。 此外，已经表明，活性依赖性蛋白质合成是高度特异性的，因为只有一小部分 的可用转录本在神经元活动后被翻译。2，3也有证据表明， 突触的合成介导可塑性，翻译中的扰动与神经系统的紊乱有关。 认知功能，如自闭症。4，5实现蛋白质合成的接近突触的网站， 传播，被翻译的转录本的特异性，以及它在疾病中的破坏，都强调了 理解转录后调控机制的性质的重要性， 神经元和突触中蛋白质的补充。 自1993年发现以来，microRNAs（miRNAs）因其广泛的功能而受到人们的重视 作为蛋白质合成的转录后调节因子，通过翻译抑制和转录不稳定6。 miRNAs调节神经元可塑性和树突棘形态发生，涉及高级脑 功能，如记忆和认知功能障碍，以及参与miRNA生物发生和功能的蛋白质 在神经元中发现，包括突触附近。1进化上保守的let-7家族的miRNA 在许多生长相关过程中作为转录后基因调控的关键介质出现，包括 发育时序（C. elegans 7和D. melanogaster 8，9）、体轴编程（M.肌肉代谢 （M. musculus 11）和癌（M. musculus 12和H. sapiens 13）. let-7家族的miRNAs是高度丰富的 在成熟分化的神经元中，我们实验室和其他实验室的工作表明let-7 miRNA水平可以 受神经元活性调节3，14-16，在缺乏脆性X智力低下的小鼠模型中被破坏 然而，明确确定mRNA全基因组同一性的方法 let-7和其他miRNAs的靶点直到最近才成为可能。本项目采用了修改后的版本 最近开发的CLEAR-CLIP技术17涉及交联和免疫沉淀， 通过与Argonaute结合的内源RNA的分子间连接和高通量测序（CIMERA- seq）。CIMERA-seq将允许关键的miRNA靶点和控制蛋白质的miRNA调控机制 在哺乳动物中枢神经系统的合成进行了详细的探讨。该提案的假设是， 在FMRP缺陷的脑中在体外和体内观察到的let-7 miRNA水平将产生miRNA的变化。 靶向分布与在神经细胞中观察到的改变的神经元可塑性、突触过度生长和蛋白质合成一致。 缺乏FMRP的小鼠模型。