miRNAs at the C. elegans neuromuscular junction: potential SMA modifiers

线虫神经肌肉接头处的 miRNA：潜在的 SMA 修饰剂

• 批准号: 8908383
• 负责人: Patrick OHern
• 金额: $ 4.31万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8908383
• 关键词: 3' Untranslated Regions; Affect; Aldicarb; American; Animal Model; Animals; Behavioral Genetics; Binding; Biogenesis; Bioinformatics; Biological Assay; Boxing; Caenorhabditis elegans; Cessation of life; Complex; DNA Sequence Alteration; Data; Defect; Disease; Fluorescence; Gemin3; Genetic; Genetic Epistasis; Hela Cells; Knowledge; Lead; Link; MEL Gene; Messenger RNA; Methods; MicroRNAs; Modeling; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Orthologous Gene; Pathway interactions; Play; Proteins; Public Health; Pump; RNA; RNA Helicase; RNA Interference; Regulation; Reporter; Role; SMN protein (spinal muscular atrophy); SMN1 gene; Signal Transduction; Spinal Muscular Atrophy; Synapses; Synaptic Transmission; Testing; Tissues; United States; Untranslated RNA; Work; base; cholinergic; design; disease-causing mutation; human DICER1 protein; infant death; knock-down; loss of function; motor neuron degeneration; mouse model; mutant; mutation carrier; nervous system disorder; neuron loss; overexpression; protein expression; public health relevance; research study; screening; synaptic function; tool

 DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA) is a neurological disorder characterized by loss of lower motor neurons. This degeneration is caused by genetic mutations that lead to decreased levels of Survival of Motor Neuron (SMN) protein. It is currently unknown how SMN reduction results in neuronal death, however recent evidence suggests that miRNA disruption may play a role. miRNAs are small non-coding RNAs predicted to regulate protein expression of a vast number of mRNAs. The RNA helicase Gemin3 pulls down with both SMN and numerous miRNAs in cultured mouse motor neurons. I hypothesize that Gemin3-associated miRNAs are misregulated in smn-1 loss-of-function(lf) animals leading to NMJ defects. smn-1(ok355) animals are defective on aldicarb, have reduced pumping and altered levels of synaptic proteins, suggesting neuromuscular junction (NMJ) defects. My preliminary data shows that loss of the C. elegans Gemin3 ortholog, mel-46, results in similar NMJ defects. Using tissue-specific rescue analysis and genetic epistasis, I will show that mel-46 is downstream of smn-1 in a pathway influencing NMJ signaling. Additionally, I have identified C. elegans orthologs of Gemin3-associated miRNAs that are necessary for proper NMJ synaptic transmission similar to smn-1 and mel-46. I will compile lists of potential mRNA targets for these candidate miRNAs using online bioinformatics tools; assigning priority to conserved targets with known synaptic function. For each potential mRNA target, I will confirm miRNA regulation and investigate whether miRNA function is altered in smn-1 lf and mel-46 lf. Furthermore, I have designed experiments to show loss of miRNA function in smn-1 lf animals is caused by mel-46 reduction. Using motor neurons from a SMA mouse model, I will show that the relationship between SMN, Gemin3 and miRNA function is conserved across species. These experiments will advance our knowledge of how SMN protein contributes to essential neuronal functions and expand our understanding of how miRNA misregulation may contribute to neurodegeneration.

 描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种以下运动神经元丧失为特征的神经系统疾病。这种变性是由基因突变引起的，基因突变会导致运动神经元存活 (SMN) 蛋白水平降低。目前尚不清楚 SMN 减少如何导致神经元死亡，但最近的证据表明 miRNA 破坏可能发挥了作用。 miRNA 是小型非编码 RNA，预计可调节大量 mRNA 的蛋白质表达。 RNA 解旋酶 Gemin3 可以在培养的小鼠运动神经元中同时抑制 SMN 和大量 miRNA。我推测 Gemin3 相关 miRNA 在 smn-1 功能丧失 (lf) 动物中受到错误调节，导致 NMJ 缺陷。 smn-1(ok355) 动物在涕灭威方面有缺陷，泵血减少并改变了突触蛋白的水平，表明神经肌肉接头 (NMJ) 缺陷。我的初步数据表明，线虫 Gemin3 直系同源基因 mel-46 的丢失会导致类似的 NMJ 缺陷。使用组织特异性救援分析和遗传上位性，我将证明 mel-46 是 smn-1 下游影响 NMJ 信号传导的途径。此外，我还鉴定出了 Gemin3 相关 miRNA 的线虫直系同源物，这些 miRNA 是正确的 NMJ 突触传递所必需的，类似于 smn-1 和 mel-46。我将使用在线生物信息学工具为这些候选 miRNA 编制潜在 mRNA 靶标列表；优先考虑具有已知突触功能的保守目标。对于每个潜在的 mRNA 靶标，我将确认 miRNA 调控并研究 smn-1 lf 和 mel-46 lf 中 miRNA 功能是否发生改变。此外，我设计的实验表明 smn-1 lf 动物中 miRNA 功能的丧失是由 mel-46 减少引起的。使用 SMA 小鼠模型的运动神经元，我将证明 SMN、Gemin3 和 miRNA 功能之间的关系在物种间是保守的。这些实验将增进我们对 SMN 蛋白如何促进基本神经元功能的了解，并扩大我们对 miRNA 失调如何导致神经退行性变的理解。