Uncovering polyadenylation pathways in neuronal signaling

揭示神经信号传导中的聚腺苷酸化途径

• 批准号: 8341254
• 负责人: SAMIE R JAFFREY
• 金额: $ 36.97万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8341254
• 关键词: Accounting; Actins; Address; Adenosine; Affect; Afferent Neurons; Antibodies; Axon; Clinical; Cues; Defect; Employee Strikes; Enzymes; Event; Exhibits; F-Actin; Gene Expression Profile; Genetic Translation; Goals; Growth Cones; Knock-out; Label; Lead; Link; Mediating; Messenger RNA; Methods; Morphology; Mutation; Natural regeneration; Nerve Growth Factors; Neurodevelopmental Disorder; Neurons; Nucleotides; Oocytes; PC12 Cells; Pathway interactions; Poly A; Polyadenylation; Polyadenylation Pathway; Polymerase; Process; Protein Biosynthesis; Protein Isoforms; Proteins; RNA Processing; RNA Splicing; Receptor Activation; Regulation; Research; Role; Screening procedure; Semaphorin-3A; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Stimulus; Synaptic plasticity; Tail; Techniques; Testing; Time; Transcript; Translating; Translational Regulation; Translations; Xenopus oocyte; analog; axon growth; axon guidance; axonal guidance; chemical genetics; in vivo; nervous system disorder; neurotrophic factor; novel; oocyte maturation; polyadenylated messenger RNA; protein expression; research study; response; Accounting; Actins; Address; Adenosine; Affect; Afferent Neurons; Antibodies; Axon; Clinical; Cues; Defect; Employee Strikes; Enzymes; Event; Exhibits; F-Actin; Gene Expression Profile; Genetic Translation; Goals; Growth Cones; Knock-out; Label; Lead; Link; Mediating; Messenger RNA; Methods; Morphology; Mutation; Natural regeneration; Nerve Growth Factors; Neurodevelopmental Disorder; Neurons; Nucleotides; Oocytes; PC12 Cells; Pathway interactions; Poly A; Polyadenylation; Polyadenylation Pathway; Polymerase; Process; Protein Biosynthesis; Protein Isoforms; Proteins; RNA Processing; RNA Splicing; Receptor Activation; Regulation; Research; Role; Screening procedure; Semaphorin-3A; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Stimulus; Synaptic plasticity; Tail; Techniques; Testing; Time; Transcript; Translating; Translational Regulation; Translations; Xenopus oocyte; analog; axon growth; axon guidance; axonal guidance; chemical genetics; in vivo; nervous system disorder; neurotrophic factor; novel; oocyte maturation; polyadenylated messenger RNA; protein expression; research study; response

DESCRIPTION (provided by applicant): The objective of the proposed research is to identify and characterize signaling mechanisms that lead to local mRNA translation in growth cones and developing axons. Studies of local translation have been hampered because the identity of specific mRNAs that are regulated by guidance cues are unknown. Furthermore the signaling mechanisms that couple receptor activation to mRNA translation are poorly understood. One mechanism to regulate local translation is polyadenylation of axonally localized transcripts, but this process is highly difficult to study. We have developed a chemical genetic approach to selectively label and recover transcripts that become polyadenylated. To do this, we synthesized a novel adenosine nucleotide that can be incorporated into polyA tails by cellular PAPs. Using this analog, we developed a deep sequencing approach to profile polyadenylation across the transcriptome. We have also sought to identify the polyA polymerase (PAP) that catalyzes polyadenylation in response to axonal signaling. By screening axons using antibodies to known PAPs, we identified a specific PAP isoform that is enriched in axons and colocalizes with actin in growth cones. As part of our overall goal to identify the intracellular signaling pathways that regulate the translation of mRNAs in axons and growth cones, the specific aims of this proposal are: (1) To profile polyadenylation events that mediate the effects of nerve growth factor (NGF) and Semaphorin 3A (Sema3A). In this aim, we will selectively label, identify, and test the functional roles of mRNAs polyadenylated in response to NGF and Sema3A in sensory neurons. (2) To identify polyadenylation and deadenylation pathways that regulate local translation in axons. In this aim, we use the polyadenylation tagging approach to identify the network of polyadenylation events induced by NGF and Sema3A within axons. Furthermore, we explore the role of deadenylation in repressing axonal translation, and will identify the axonal deadenylase that regulates axonal mRNA translation. (3) To establish the PAPs that regulate intra-axonal translation. In this aim, we characterize a novel axonally enriched PAP, and determine its roles in local translation in axons. These studies will characterize the role of a novel axonal PAP, potentially uncovering the "missing" regulator of polyadenylation in axons. The experiments proposed here provide a comprehensive analysis of polyadenylation networks that are elicited by axonal guidance cues, and will identify new proteins that regulate polyadenylation in axons. Since polyadenylation is a major mechanism controlling protein expression, we anticipate that the methods and translational regulators that will be characterized here will have broad relevance to signaling pathways in diverse cellular contexts. PUBLIC HEALTH RELEVANCE: Regulated protein synthesis is important for synaptic plasticity, axon guidance, and regeneration, and mutations in proteins that regulate protein synthesis or RNA processing have been linked to numerous neurological disorders. Although polyadenylation of mRNA is known to be a major mechanism by which axon guidance cues and neurotrophins activate protein synthesis, methods to explore polyadenylation on a transcriptome-wide scale are not available, and the enzymes that mediate polyadenylation are poorly understood. This project will, for the first time, identify the polyadenylation pathways induced in response to neuronal stimuli, elucidate the signaling pathways that regulate the polyadenylation pathways that control local translation in axons, and identify a novel enzyme regulator of polyadenylation that may have critical roles in local translation pathways that influence axon pathfinding and synaptic plasticity.

描述（由申请人提供）：拟议的研究的目的是识别和表征导致生长锥和发育轴突局部mRNA翻译的信号传导机制。局部翻译的研究受到了阻碍，因为由指导提示调节的特定mRNA的身份尚不清楚。此外，对夫妇受体激活向mRNA翻译的信号传导机制也很少了解。调节局部翻译的一种机制是轴突局部转录本的聚腺苷酸化，但是该过程很难研究。我们已经开发了一种化学遗传学方法，以选择性地标记和恢复成为聚腺苷酸化的转录本。为此，我们合成了一种新型的腺苷核苷酸，该核苷酸可以通过细胞PAPS掺入Polya尾巴。使用此类似物，我们开发了一种深层测序方法来跨转录组谱图聚腺苷酸化。我们还试图鉴定多A聚合酶（PAP），该聚合酶响应轴突信号传导催化聚腺苷酸化。通过使用抗体对已知PAP的抗体筛选轴突，我们确定了一种特定的PAP同工型，该PAP同工型富含轴突并与生长锥中的肌动蛋白共定位。作为我们确定调节轴突和生长锥体中mRNA翻译的细胞内信号传导途径的总体目标的一部分，该提案的具体目的是：（1）介导神经生长因子（NGF）和Semaphorin 3A（SEMA3A）的影响的剖面多腺苷酸化事件。在此目标中，我们将在感觉神经元中选择性地标记，识别和测试对NGF和SEMA3A的mRNA聚腺苷酸化的功能作用。 （2）确定调节轴突中局部翻译的聚腺苷酸化和去甲基化途径。在此目标中，我们使用聚腺苷酸化标记方法来识别NGF和SEMA3A在轴突中诱导的聚腺苷酸化事件网络。此外，我们探讨了deadenyration在抑制轴突翻译中的作用，并将识别调节轴突mRNA翻译的轴突降苯酶。 （3）建立调节轴内翻译的PAP。在此目标中，我们表征了一种新型的轴突富集的PAP，并确定其在轴突中局部翻译中的作用。这些研究将表征新型的轴突PAP的作用，可能会发现轴突中多腺苷酸化的“缺失”调节剂。这里提出的实验提供了对轴突引导提示引起的聚腺苷酸化网络的全面分析，并将确定调节轴突中聚腺苷酸化的新蛋白质。由于聚腺苷酸化是控制蛋白质表达的主要机制，因此我们预计此处将表征的方法和翻译调节剂在不同的细胞环境下将与信号通路具有广泛的相关性。 公共卫生相关性：受调节的蛋白质合成对于调节蛋白质合成或RNA处理的蛋白质的突触可塑性，引导和再生以及突变与多种神经系统疾病有关。尽管已知MRNA的聚腺苷酸化是一种主要机制，轴突引导线索和神经营养蛋白激活蛋白质的合成，但无法在整个转录组范围内探索多腺苷酸化的方法，并且介导的聚烯基化的酶是众所周知的。该项目将首次确定响应神经元刺激诱导的多腺苷酸化途径，阐明控制轴突中局部翻译的聚腺苷酸化途径的信号传导途径，并确定一种新型的酶调节剂，这些酶调节剂在多腺苷酸化中，该酶调节剂可能在局部翻译道路中具有关键的轴突途径，从而影响轴突的途径和轴突固定型固定速度。