High-affinity RNA targets of Survival Motor Neuron Protein

描述(申请人提供)：脊髓性肌萎缩症(SMA)是一种发育障碍，其特征是儿童早期运动神经元进行性丧失。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。多数SMA与SMN1基因缺失或突变引起的SMN蛋白表达降低有关。该基因的一个几乎相同的拷贝SMN1不能补偿SMN1的缺失，这是由于SMN2的主要外显子7跳过，从而产生截短蛋白SMN？7。SMN的Tudor结构域中的一个错义突变(E134K)也与SMA有关。在许多重要功能中，SMN参与了小核核糖核蛋白的生物发生、转录、前mRNA剪接、大分子转运、信号转导和应激颗粒的形成。SMN含有一个独特的核酸结合域，在体外已被证明对聚鸟苷(Poly-Rg)RNA有偏好。我们最近结束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本(RNA)直接相互作用发挥更广泛的作用。在这里，我们将使用紫外线交联和免疫沉淀(CLIP)和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的分析。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的转录范围内的相互作用。我们将通过改变各种参数来优化紫外光交联条件，包括使用PAR-CLIP(光活化核糖核苷增强的CLIP)中的光反应性核糖核苷。我们将使用高通量测序来分析与SMN相关的片段标签(交联序列)。为了分析那些不能在CLIP/PAR-CLIP中扩增的序列，我们将使用iCLIP(单个核苷酸分辨率UV-CLIP)。为了确定细胞RNA与SMN相互作用的性质，我们将CLIP标签映射到人类基因组。利用基因组图谱，我们将确定交联链诱导突变位点(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，我们将验证由CLIP标签和CIMS数据揭示的新的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，我们将评估在SMN水平降低时SMN在转录水平上的相互作用的变化。这项研究的结果将揭示SMN相互作用转录本的特征，这些转录本在SMN浓度降低时会发生剧烈变化。为了评估与疾病相关的突变SMN蛋白与野生型SMN的转录水平的相互作用，我们将用SMN？7和E134K进行CLIP/PAR-CLIP/iCLIP实验。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，我们将用报告分析进行基于细胞的实验。此外，我们还将 验证我们在对照组和SMA小鼠的运动神经元上进行的CLIP实验的关键结果。我们的建议有可能发现新的SMN功能，对更好地理解SMA发病的分子机制具有重要意义。) 公共卫生相关性：发育过程中缺乏存活运动神经元(SMN)蛋白会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之一。在这个提案中，我们将对RNA-SMN相互作用进行全局分析，以确定新的SMN功能，这对更好地理解SMA发病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. ) PUBLIC HEALTH RELEVANCE: Deficiency of Survival Motor Neuron (SMN) protein during development causes spinal muscular atrophy (SMA), one of the leading genetic causes of infant mortality. In this proposal, we will perform global analysis of RNA-SMN interactions to identify novel SMN functions with significance to a better understanding of molecular mechanism of SMA pathogenesis.)