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）是一种以下肢运动神经元丧失为特征的神经系统疾病。这种退化是由导致运动神经元（SMN）存活蛋白水平下降的基因突变引起的。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。mirna是一种小的非编码rna，预计可以调节大量mrna的蛋白质表达。RNA解旋酶Gemin3在培养的小鼠运动神经元中与SMN和许多mirna一起下调。我假设在smn-1功能丧失（lf）动物中，gemin3相关的mirna被错误调控，导致NMJ缺陷。smn-1（ok355）动物对涕灭威有缺陷，泵送减少，突触蛋白水平改变，提示神经肌肉连接（NMJ）缺陷。我的初步数据显示，秀丽隐杆线虫Gemin3同源基因mel-46的缺失，会导致类似的NMJ缺陷。通过组织特异性拯救分析和遗传上位，我将证明mel-46在影响NMJ信号通路中位于smn-1的下游。此外，我已经鉴定出秀丽隐杆线虫与gemin3相关的mirna同源物，这些mirna与smn-1和mel-46类似，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编制这些候选mirna的潜在mRNA靶标列表；优先考虑具有已知突触功能的保守靶点。对于每个潜在的mRNA靶点，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，我设计了实验来证明smn-1动物中miRNA功能的丧失是由mel-46减少引起的。使用来自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中是保守的。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，并扩大我们对miRNA失调如何导致神经变性的理解。

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.