Characterization of a complex regulatory element of Spinal Muscular Atrophy genes

描述(申请人提供)：人类有两个几乎相同的存活运动神经元(SMN)基因，SMN1和SMN2。由于SMN1缺失和/或突变导致的SMN水平低导致脊髓性肌萎缩症(SMA)，这是导致婴儿死亡的主要遗传原因。SMN1不能补偿由于外显子7第6位C到T突变(转录本中的C6U)造成的SMN1的丢失。C6U削弱了32-剪接位点并触发SMN2外显子7的跳过，导致合成截断的蛋白质(SMN？7)，这是不稳定的。众所周知，旨在纠正SMN2外显子7剪接的策略有望治愈。这一建议源于我们最近发现的一种独特的RNA结构，该结构是由长距离相互作用形成的，作为SMN2外显子7剪接的调节因子(Singh等人，核酸研究，2013年，DOI：10.1093/NAR/GKT609)。我们将这种结构称为内茎到LDI-1(ISTL1)。采用SHAPE(选择性22-羟基酰化分析)方法，证实了ISTL1的形成及其功能意义。我们证明了反义寡核苷酸(ASO)介导的ISTL1 32链的隔离完全纠正了SMN2外显子7的剪接，并恢复了SMA患者细胞中SMN和SMN相互作用蛋白Gin2的高水平。我们的结果还显示，ISTL1的32条链位于一个大的 抑制区，我们称之为内含子剪接抑制子N2(ISS-N2)。为了继续我们的领导，这里我们建议表征额外的(新的)内含子顺式元件及其在调节SMN2外显子7剪接中的同源交易因子。该提案的一部分旨在验证ISS-N2靶向ASOS的治疗效果。在目标1中，我们将使用重叠缺失、基于ASO的策略和形状分析来确定SMN2内含子7中新的顺式元件的重要性。我们将在不同类型的细胞中验证我们的发现，包括 运动神经元样NSC34细胞。我们将研究更大的结构背景对SMN2外显子7剪接位点可及性的影响。我们将确定SMN1特定的突变是否导致SMN1和SMN2前mRNAs之间的结构差异，特别是在SMN外显子7的剪接位点。此外，我们还将评估SMN中的关键顺式元件 内含子6影响内含子7的结构背景，并可能导致52内含子的重塑 在目标2中，我们将使用过度表达和基于siRNA的策略来识别有助于ISTL1形成和/或将ISTL1用作自我招募的剪接因子。我们将使用生物素化的寡核苷酸作为捕获装置来捕获沉积在SMN2内含子7上的新的RNA-蛋白质复合体，这些复合体对LDI的抑制作用至关重要。我们以前已经证明TIA1通过与内含子7结合来刺激SMN2外显子7的剪接。我们还证明了TIA1的富含Q的结构域在调节SMN2外显子7剪接中的关键作用。最近，TIA1富Q结构域中的一个点突变被证明可以导致韦兰德远端肌病，并促进SMN2外显子7的跳跃。基于这些发现和我们的初步结果，我们将研究另一个含有剪接调控因子的富含Q的结构域SFPQ在SMN2外显子7剪接中的作用。我们将使用UV交联法、足迹法、体外结合法和基于形状的方法来表征RNA-蛋白质的相互作用。在目标3中，我们将在轻度和重度SMA小鼠模型上进行体内研究，以确定ISS-N2靶向磷二酸铅吗啉齐聚物(PMO)的治疗效果。我们将根据最近报道的几项成功的体内研究设计我们的实验计划，并将采用严格的样本量估计、随机化和盲法标准。成功的结果将导致开发一种基于ASO的治疗SMA的新方法。

DESCRIPTION (provided by applicant): Humans have two nearly identical copies of Survival Motor Neuron (SMN) gene, SMN1 and SMN2. Low SMN levels due to deletion and/or mutation of SMN1 lead to spinal muscular atrophy (SMA), a major genetic cause of infant mortality. SMN2 fails to compensate for the loss of SMN1 due to a C to T mutation at the 6th position (C6U in transcript) in exon 7. C6U weakens the 32-splice site and triggers SMN2 exon 7 skipping, resulting in synthesis of a truncated protein (SMN¿7), which is unstable. It is known that strategies aimed at correction of SMN2 exon 7 splicing hold the promise for a cure. This proposal emanates from our recent discovery of a unique RNA structure formed by a long-distance interaction (LDI) as a regulator of SMN2 exon 7 splicing (Singh et al., Nucleic Acids Res., 2013, doi:10.1093/nar/gkt609). We call this structure internal stem through LDI-1 (ISTL1). Employing the SHAPE (Selective 22-Hydroxyl Acylation analyzed by Primer Extension) method, we confirmed the formation and functional significance of ISTL1. We showed that an antisense oligonucleotide (ASO)-mediated sequestration of the 32 strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, an SMN-interacting protein, in SMA patient cells. Our results also revealed that the 32 strand of ISTL1 is located within a large inhibitory region that we termed intronic splicing silencer N2 (ISS-N2). To continue with our lead, here we propose to characterize additional (novel) intronic cis-elements and their cognate transacting factors in regulation of SMN2 exon 7 splicing. A part of the proposal is aimed at validating the therapeutic efficacy of ISS-N2- targeting ASOs. In Aim 1, we will use overlapping deletions, ASO-based strategies and SHAPE analyses to determine the significance of novel cis-elements within SMN2 intron 7. We will validate our findings in different cell types including motor neuron-like NSC34 cells. We will examine the effect of a larger structural context on the accessibility of the splice sites of SMN2 exon 7. We will determine whether SMN2-specific mutations lead to a structural difference between SMN1 and SMN2 pre-mRNAs, particularly at the splice sites of SMN exon 7. In addition, we will evaluate whether critical cis-elements within intron 6 affect the structural context of intron 7 and potentially lead to the remodeling of the 52 slice site of exon 7. In Aim 2, we will employ over-expression and siRNA-based strategies to identify splicing factors that assist ISTL1 formation and/or use ISTL1 as a site for self-recruitment. We will use a biotinylated oligonucleotide as a trapping device to capture novel RNA-protein complexes that are deposited on SMN2 intron 7 and are critical for the inhibitory effect of LDI. We have previously shown that TIA1 stimulates SMN2 exon 7 splicing by binding to intron 7. We also demonstrated critical role of the Q-rich domain of TIA1 in regulation of SMN2 exon 7 splicing. Recently, a point mutation within Q-rich domain of TIA1 has been shown to cause Welander distal myopathy as well as promote SMN2 exon 7 skipping. Based on these findings and our preliminary results, we will examine the role of another Q-rich domain containing splicing regulator, SFPQ, in SMN2 exon 7 splicing. We will employ UV-crosslinking, footprinting, in vitro binding and SHAPE-based approaches to characterize RNA-protein interactions. In Aim 3, we will perform in vivo studies in a mild as well as in a severe mouse model of SMA to determine the therapeutic efficacy of an ISS-N2 targeting lead phosphorodiamidate morpholino oligomer (PMO). We will design our experimental plan based on several successful in vivo studies reported recently and will employ rigorous criteria of sample-size estimation, randomization and blinding. A successful outcome will lead to the development of a novel ASO-based therapy for SMA.