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，是最常见的遗传疾病 婴儿死亡原因。SMA是由于存活运动神经元(SMN)蛋白表达不足所致。 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基因，并增加SMN蛋白。然而，这种方法的治疗性救援可能会受到 一个人拥有的SMN2拷贝数。因为患有最常见和最严重的SMA的人 如果只有1到2个SMN2拷贝，他们可能需要其他治疗策略。最近，第一个长非编码 报道了与SMN2基因相关的RNA(LncRNA)。这种名为SMN-AS1的lncRNA招募了一名 表观遗传抑制复合体，称为多梳抑制复合体2(PRC2)，与SMN2启动子和 抑制SMN2转录。反义寡核苷酸对SMN-AS1基因的敲除作用 增加细胞培养模型中SMN的表达，联合应用对SMA小鼠的行为挽救作用不大 SMN2剪接矫正药。LncRNAs具有广泛的二级结构和本地域 为他们的职能做出贡献。目前尚不清楚SMN-AS1的结构和结构域如何影响其 SM2的表观遗传抑制。SMN-AS1的SMN-AS1结构域的阐明和阻断 使用改良的阻断ASO的抑制可能有助于推进靶向SMN-AS1作为治疗的治疗 SMA的。 该建议旨在发现SMN-AS1的各个功能结构域，并了解SMN-AS1是如何 与SMN2启动子和PRC2相互作用，调节SMN的表达。SA#1将采用简化的 利用荧光素酶报告策略确定抑制SMN2所需的SMN-AS1结构域。比较 SMN-AS1的全长或截短形式过表达后的SMN表达将有助于缩小对 文字记录的关键功能区。SA#2中的实验将确定是否使用 阻断靶向SMN-AS1的ASO改变原代培养小鼠皮质SMN RNA或蛋白的表达 神经元。最后，SA#3将阐明SA#2中阻止的结构域是否需要SMN-AS1绑定 与小鼠原代神经元中的SMN2启动子或PRC2结合。这些实验使用先进的技术来 研究RNA：DNA和RNA：蛋白质的相互作用。此外，无偏见的RNA下拉紧随其后的质量 光谱分析将识别与SMN-AS1结合的新蛋白质，为研究其他潜在的可能性打开大门 SMN-AS1的作用机制。这些实验将使我们深入了解lncRNAs如何调控基因。 表达，并将促进更好的ASO设计，有效地将SMN-AS1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.