High-affinity RNA targets of Survival Motor Neuron Protein

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种发育障碍，其特征为儿童早期运动神经元的进行性丧失。根据发生频率，SMA被列为婴儿死亡的第二大遗传原因，仅次于囊性纤维化。大多数SMA病例与运动神经元1（SMN1）基因缺失或突变导致的SMN蛋白水平降低有关。一个几乎相同的拷贝的基因，SMN2，未能弥补损失的SMN1由于占主导地位的SMN2外显子7跳跃，产生截短的蛋白质，SMN？7. SMN的tudor结构域中的单个错义突变（E134K）也与SMA相关。在几个重要的功能中，SMN已经涉及小核核糖核蛋白（snRNP）的生物发生、转录、前mRNA剪接、大分子运输、信号转导和应激颗粒形成。SMN含有一个独特的核酸结合结构域，其已被证明在体外对聚鸟苷（poly rG）RNA具有偏好性。我们最近完成了一项体外选择实验，揭示了SMN识别的序列基序的多样性。这些结果支持SMN通过与各种细胞转录物（RNA）直接相互作用发挥更广泛的作用。在这里，我们将使用UV交联和免疫沉淀（CLIP）以及高通量测序的强大方法对SMN的转录组范围内的相互作用进行系统分析。在目的1中，我们将进行CLIP实验，以捕获神经元SH-SY5Y细胞中SMN的转录组范围的相互作用。我们将通过修改各种参数来优化UV交联条件，包括使用PAR-CLIP（光活化核糖核苷增强型CLIP）中的光反应性核糖核苷。我们将采用高通量测序来分析与SMN相关的CLIP标签（交联序列）。为了分析那些在CLIP/PAR-CLIP中不可扩增的序列，我们将采用iCLIP（单个核苷酸分辨率UV-CLIP）。为了确定与SMN相互作用的细胞RNA的性质，我们将CLIP标签映射到人类基因组。使用基因组图谱，我们将确定交联诱导突变位点（CIMS），这将有助于确定负责SMN相互作用的基序与单核苷酸的精度。在目标2中，我们将验证CLIP标签和CIMS数据揭示的新型RNA-SMN相互作用的功能意义。SMA的严重程度受SMN水平的影响（SMN水平越低，严重程度越高）。因此，我们将评估SMN水平降低时SMN全转录组相互作用的变化。这项研究的结果将揭示SMN相互作用的转录本的签名，这些转录本在SMN浓度降低时发生了巨大变化。为了评估疾病相关突变SMN蛋白的转录组范围的相互作用与野生型SMN不同，我们将用SMN7和E134 K进行CLIP/PAR-CLIP/iCLIP实验。为了揭示RNA-SMN相互作用影响特定转录物的剪接、稳定性和运输的可能机制，我们将使用报告基因测定进行基于细胞的实验。我们亦会 验证我们在从对照和SMA小鼠获得的运动神经元中的CLIP实验的关键发现。我们的提议有可能识别新型SMN功能，这对于更好地了解SMA发病机制的分子机制具有重要意义。)

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a developmental disorder characterized by a progressive loss of motor neurons during early childhood. Based on the frequency of occurrence, SMA is ranked as the second leading genetic cause of infant mortality after cystic fibrosis. Most cases of SMA are associated with the low levels of SMN protein due to deletion or mutation of Survival Motor Neuron 1 (SMN1) gene. A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 owing to predominant SMN2 exon 7 skipping that produces a truncated protein, SMN?7. A single missense mutation (E134K) in tudor domain of SMN has been also linked to SMA. Among several important functions, SMN has been implicated in biogenesis of small-nuclear ribonucleoproteins (snRNPs), transcription, pre-mRNA splicing, macromolecular trafficking, signal transduction and stress granule formation. SMN contains a distinct nucleic acid binding domain that has been shown to have preference for poly-guanosine (poly rG) RNAs in vitro. We have recently concluded an in vitro selection experiment that revealed diversity of sequence motifs recognized by SMN. These results support a wider role of SMN through direct interactions with a variety of cellular transcripts (RNAs). Here we will perform a systematic analysis of transcriptome-wide interactions of SMN using powerful approaches of UV crosslinking and immunoprecipitation (CLIP) and high throughput sequencing. In Aim 1, we will perform CLIP experiments to capture transcriptome-wide interactions of SMN in neuronal SH-SY5Y cells. We will optimize UV-crosslinking conditions by modifying various parameters, including use of photoreactive ribonucleosides as in PAR-CLIP (Photoactivatable- Ribonucleoside-Enhanced CLIP). We will employ high throughput sequencing to analyze CLIP tags (crosslinked sequences) associated with SMN. To analyze those sequences that are not amplifiable in CLIP/PAR-CLIP, we will employ iCLIP (individual nucleotide resolution UV-CLIP). To determine the nature of cellular RNAs interacting with SMN, we will map CLIP tags to human genome. Using genomic mapping, we will determine crosslink-induced mutation sites (CIMS) that will help identify motifs responsible for SMN interaction with a single-nucleotide precision. In aim 2, we will validate the functional significance of novel RNA-SMN interactions revealed by CLIP tags and CIMS data. Severity of SMA is affected by level of SMN (lower the SMN levels higher the severity). Therefore, we will assess the alterations in the transcriptome-wide interactions of SMN at reduced levels of SMN. Findings of this study will reveal signature of SMN-interacting transcripts that are drastically altered at reduced SMN concentrations. To assess that the transcriptome-wide interactions of disease-associated mutant SMN proteins are distinct from the wild type SMN, we will perform CLIP/PAR-CLIP/iCLIP experiments with SMN¿7 and E134K. To uncover the possible mechanism by which RNA-SMN interactions affect splicing, stability and trafficking of specific transcripts, we will perform cell-based experiments with reporter assays. Also, we will validate the key findings of our CLIP experiments in motor neurons obtained from control and SMA mice. Our proposal has potential to identify novel SMN functions with significance to a better understanding of molecular mechanism of SMA pathogenesis. )