High-affinity RNA targets of Survival Motor Neuron Protein

运动神经元存活蛋白的高亲和力 RNA 靶标

• 批准号: 8532065
• 负责人: RAVINDRA N SINGH
• 金额: $ 20.93万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532065
• 关键词: Affect; Affinity; Amino Acids; Biogenesis; Biological Assay; C-terminal; Cells; Complex; Cystic Fibrosis; Cytoplasmic Granules; Data; Development; Disease; Exons; Frequencies; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genomics; Guanosine; Human Genome; Immunoprecipitation; In Vitro; Individual; Induced Mutation; Infant Mortality; Length; Life; Link; Maps; Missense Mutation; Molecular; Motor Neurons; Mus; Mutation; Nature; Neurodegenerative Disorders; Neurons; Nucleic Acid Binding; Nucleotides; Outcome; Pathogenesis; Post-Translational Protein Processing; Proteins; RNA; RNA Splicing; RNA analysis; RNA-Binding Proteins; Reporter; Resolution; Ribonucleosides; Role; SMN protein (spinal muscular atrophy); SMN2 gene; Severities; Signal Transduction; Site; Small Nuclear Ribonucleoproteins; Spinal Muscular Atrophy; Stress; Techniques; Testing; Transcript; base; cell type; crosslink; developmental disease; early childhood; mRNA Precursor; motor neuron function; mutant; novel; preference; protein protein interaction; research study; trafficking

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. )

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种发育障碍，其特征是幼儿期运动神经元逐渐丧失。根据发生频率，SMA 被列为继囊性肌萎缩症之后婴儿死亡的第二大遗传原因。大多数 SMA 病例与运动神经元存活 1 (SMN1) 基因缺失或突变导致的 SMN 蛋白水平低有关，该基因几乎相同的 SMN2 拷贝无法弥补这一缺陷。由于 SMN2 外显子 7 的主要跳跃导致 SMN1 缺失，从而产生截短的蛋白 SMN?7，SMN 的 tudor 结构域中的单个错义突变 (E134K) 也与 SMA 相关。小核核糖核蛋白 (snRNP)、转录、前 mRNA 剪接、大分子运输、信号转导和应激SMN 含有一个独特的核酸结合结构域，该结构域在体外已显示出对聚鸟苷 (poly rG) RNA 的偏好。我们最近完成了一项体外选择实验，该实验揭示了 SMN 识别的序列基序的多样性。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，我们将使用强大的 UV 交联和免疫沉淀 (CLIP) 方法对 SMN 的转录组范围内的相互作用进行系统分析。在目标 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- 中的光反应性核糖核苷。 CLIP（光激活核糖核苷增强 CLIP）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 中无法扩增的序列，我们将使用 iCLIP（单个核苷酸分辨率 UV-CLIP）来确定与 SMN 相互作用的细胞 RNA 的性质，我们将使用基因组将 CLIP 标签映射到人类基因组。通过映射，我们将确定交联诱导突变位点 (CIMS)，这将有助于以单核苷酸精度识别负责 SMN 相互作用的基序。在目标 2 中，我们将验证所揭示的新型 RNA-SMN 相互作用的功能意义。通过 CLIP 标签和 CIMS 数据，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 转录组范围内相互作用的变化。研究将揭示 SMN 相互作用转录本的特征，这些转录本在 SMN 浓度降低时会发生显着改变。为了评估疾病相关突变 SMN 蛋白的转录组范围内的相互作用与野生型 SMN 的不同，我们将进行研究。使用 SMN 进行 CLIP/PAR-CLIP/iCLIP 实验¿ 7 和 E134K。为了揭示 RNA-SMN 相互作用影响特定转录本的剪接、稳定性和运输的可能机制，我们将进行基于细胞的报告基因实验。 验证我们在对照小鼠和 SMA 小鼠运动神经元中进行的 CLIP 实验的主要发现，我们的建议有可能确定新的 SMN 功能，这对于更好地理解 SMA 发病机制具有重要意义。