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

 描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种神经系统疾病，其特征为下运动神经元缺失。这种退化是由导致运动神经元存活（SMN）蛋白水平降低的基因突变引起的。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破坏可能起作用。miRNAs是一种小的非编码RNA，预测可调节大量mRNA的蛋白质表达。RNA解旋酶Gemin 3在培养的小鼠运动神经元中与SMN和许多miRNA一起下拉。我假设Gemin 3相关的miRNAs在smn-1功能丧失（lf）动物中被错误调节，导致NMJ缺陷。smn-1（ok 355）动物在涕灭威方面有缺陷，泵送减少，突触蛋白水平改变，表明神经肌肉接头有缺陷。我的初步数据显示C. elegans Gemin 3直系同源物mel-46导致类似的NMJ缺陷。使用组织特异性拯救分析和遗传上位性，我将表明，mel-46是下游的smn-1的影响NMJ信号传导的途径。此外，我还确定了C。与smn-1和mel-46类似，Gemin 3相关的miRNA是适当的NMJ突触传递所必需的。我将使用在线生物信息学工具为这些候选miRNAs编制潜在的mRNA靶点列表;优先考虑具有已知突触功能的保守靶点。对于每一个潜在的mRNA靶点，我将确认miRNA的调控，并研究smn-1 lf和mel-46 lf中miRNA的功能是否改变。此外，我设计了实验来显示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，Gemin 3和miRNA功能之间的关系在物种间是保守的。这些实验将推进我们对SMN蛋白如何促进基本神经元功能的认识，并扩展我们对miRNA失调如何促进神经退行性变的理解。

 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.