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

神经科学中最耐人寻味和最复杂的问题之一是关于分子 突触参与学习和记忆的机制。早在20世纪60年代就开始了，一个不断增长的 大量证据表明，学习和长期记忆形成需要新的蛋白质合成。 此外，已经证明依赖活性的蛋白质合成是高度特异的，因为它只是一个很小的子集。 2，3也有证据表明，局部蛋白质 突触的合成调节可塑性，翻译上的干扰与神经功能障碍有关 认知功能，如自闭症。4，5蛋白质合成接近突触部位的实现 传播，翻译的转录本的特异性，以及它在疾病中的破坏，突显了 了解转录后调控机制的性质的重要性，这些机制塑造了 神经元和突触中的蛋白质补体。 自1993年被发现以来，microRNAs(MiRNAs)因其广泛的功能而受到重视 作为蛋白质合成的转录后调节因子，通过翻译抑制和转录失稳。 MiRNAs调节神经元可塑性和树突棘的形态发生，与高级大脑有关 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，包括近突触1.进化上保守的let-7家族的miRNAs 在许多与生长相关的过程中作为转录后基因调控的关键媒介出现，包括 发育计时(C.eleans7和D.Blackogaster8，9)、体轴编程(M.Musculus 10)、新陈代谢 (肌肉分支杆菌11)和癌症(肌肉分支杆菌12和智人分支杆菌13)。Let-7家族miRNAs含量非常丰富 在成熟分化的神经元中，我们和其他实验室的工作表明，let-7 miRNA水平可以是 受神经元活性3，14-16的调节，在缺乏脆性X智力低下的小鼠模型中被破坏 蛋白质(FMRP)。31然而，一种明确确定mRNA全基因组同一性的方法 直到最近，针对let-7和其他miRNAs的靶标才成为可能。本项目采用的是修改后的版本 在最近开发的涉及交联和免疫沉淀的透明夹层技术17中 通过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miRNA调控蛋白质的机制 在哺乳动物中枢神经系统的合成有待于非常详细的探索。这一提议的假设是降低了 在体外和体内观察到的FMRP缺陷脑中的let-7miRNA水平将产生miRNA的变化- 靶点轮廓与观察到的神经元可塑性、突触过度生长和蛋白质合成的改变一致 缺乏FMRP的小鼠模型。

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.