Uncovering the functional domains through which SMN-AS1 regulates the survival motor neuron gene

项目摘要 脊髓性肌萎缩症（SMA）是一种进行性遗传性神经肌肉疾病， 婴儿死亡的原因。SMA是由运动神经元存活蛋白（SMN）表达不足引起的。 FDA于2016年12月批准了第一种治疗SMA的药物，该药物纠正了SMA中的剪接异常。 SMN 2基因，并增加SMN蛋白。然而，通过这种方法的治疗性挽救可能受到以下因素的限制： 一个人拥有的SMN 2拷贝数。因为患有最常见和最严重的SMA的个体 如果患者只有1或2个SMN 2拷贝，则可能需要其他治疗策略。最近，第一个长非编码 报告了SMN 2基因相关RNA（lncRNA）。这种称为SMN-AS 1的lncRNA， 表观遗传抑制复合物，称为多梳抑制复合物2（PRC 2），SMN 2启动子和 抑制SMN 2转录。当使用反义寡核苷酸（ASO）敲低SMN-AS 1时， 在细胞培养模型中增加SMN表达，当与 SMN 2剪接纠正药物lncRNA具有广泛的二级结构和局部结构域， 有助于其功能。目前尚不清楚SMN-AS 1的结构和结构域如何有助于其表达。 SMN 2的表观遗传抑制。阐明和阻断SMN-AS 1中对SMN 2有贡献的结构域 使用修饰的阻断性ASO进行抑制可能有助于推进SMN-AS 1靶向治疗， 的SMA。 该提案旨在发现SMN-AS 1的各个功能域，并了解SMN-AS 1如何在细胞内发挥作用。 与SMN 2启动子和PRC 2相互作用以调节SMN表达。SA#1将采用简化的 荧光素酶报告基因策略来描绘抑制SMN 2所需的SMN-AS 1结构域。比较 SMN-AS 1全长或截短形式过表达后的SMN表达将有助于缩小SMN-AS 1表达的范围。 转录本的关键功能区域。SA#2中的实验将确定是否使用 阻断靶向SMN-AS 1的ASOs改变培养的小鼠原代皮层中SMN RNA或蛋白质表达 神经元最后，SA#3将阐明SA#2中阻断的结构域是否是SMN-AS 1结合所需的 与SMN 2启动子或PRC 2在小鼠原代神经元中的相互作用。这些实验使用先进的技术， 研究RNA：DNA和RNA：蛋白质相互作用。此外，无偏的RNA下拉，随后是质量 光谱法将确定与SMN-AS 1结合的新蛋白质，为研究其他潜在的蛋白质打开大门。 SMN-AS 1的作用机制。这些实验将使我们深入了解lncRNA如何调节基因表达。 表达，并将促进更好的阿索设计，以有效靶向SMN-AS 1作为临床前靶点， SMA的治疗

Project Summary Spinal muscular atrophy (SMA) is a progressive inherited neuromuscular disorder and the most common genetic cause of death in infants. SMA is caused by the insufficient expression of survival motor neuron (SMN) protein. The FDA approved the first drug to treat SMA in December 2016, which corrects a splicing abnormality in the SMN2 gene, and increases SMN protein. However, therapeutic rescue by this method may be limited by the number of SMN2 copies that a person has. Because individuals with the most common and severe form of SMA have only 1 or 2 copies of SMN2, they may require other therapeutic strategies. Recently, the first long noncoding RNA (lncRNA) associated with the SMN2 gene was reported. This lncRNA, called SMN-AS1, recruits an epigenetic repressive complex, called the polycomb repressive complex 2 (PRC2), to the SMN2 promoter and represses SMN2 transcription. While knockdown of SMN-AS1 using antisense oligonucleotides (ASOs) increases SMN expression in cell culture models, behavioral rescue of SMA mice is modest when combined with the SMN2 splice-correcting drug. LncRNAs have extensive secondary structure and local domains that contribute to their function. It is currently unknown how the structure and domains of SMN-AS1 contribute to its epigenetic repression of SMN2. Elucidating and blocking the domains of SMN-AS1 that contribute to SMN2 repression using modified blocking ASOs may help advance targeting SMN-AS1 as a therapeutic for treatment of SMA. This proposal aims to discover the individual functional domains of SMN-AS1 and understand how SMN-AS1 interacts with the SMN2 promoter and PRC2 to regulate SMN expression. SA#1 will employ a simplified luciferase reporter strategy to delineate domains of SMN-AS1 required for repressing SMN2. Comparison of SMN expression after overexpression of full-length or truncated forms of SMN-AS1 will help narrow in on the critical functional regions of the transcript. Experiments in SA#2 will determine if blocking different domains using blocking ASOs targeting SMN-AS1 alters SMN RNA or protein expression in cultured mouse primary cortical neurons. Finally, SA#3, will elucidate whether the domains blocked in SA#2 are required for SMN-AS1 binding to the SMN2 promoter or PRC2 in mouse primary neurons. These experiments use advanced techniques to investigate RNA:DNA and RNA:protein interactions. Furthermore, an unbiased RNA pulldown followed by mass spectrometry will identify novel proteins that bind to SMN-AS1, opening the door to investigating other potential mechanisms of action of SMN-AS1. These experiments will give insight into how lncRNAs may regulate gene expression and will facilitate better ASO design for targeting SMN-AS1 effectively as a preclinical target for treatment of SMA.