Determinants that regulate splicing of SMN

调节 SMN 剪接的决定因素

• 批准号: 6331892
• 负责人: Christian L. Lorson
• 金额: $ 22.88万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-15 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6331892
• 关键词: RNA splicing; animal genetic material tag; binding sites; disease /disorder model; gene expression; gene induction /repression; gene mutation; genetic enhancer element; genetic regulatory element; introns; mutant; nucleic acid sequence; precursor mRNA; progressive spinal muscular atrophy; transcription factor

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) results from the loss of both telomeric 5q13 copies of the SMN1 gene. The centromeric 5q13 SMN2 gene encodes an identical protein. While the loss of SMN2 does not lead to the development of SMA, the presence of SMN2 acts as a disease modifier in a dose-dependent manner in the absence of SMN1. The SMN1 gene produces a full-length transcript, while the primary product of SMN2 is an exon-skipped mRNA lacking exon 7. We reported that a single non-polymorphic nucleotide difference between SMN1 and SMN2 is the basis for this alternative splicing. By comparing hybrid SMN genes derived from SMA patients with unaffected relatives, we demonstrated that the origin of this nucleotide (SMN1 or SMN2) dictated clinical outcome. Therefore, the clinical evolution of SMA correlates with the splicing of SMN genes. SMN protein self-associates, and the protein translated from the alternative spliced SMN2 RNA, has a reduced ability to form oligomers and is less-stable compared to the full-length protein. Furthermore, SMN point mutants from SMA patients show defective oligomerization proportional to their disease severity. These studies have defined the genetic and biochemical basis for development of SMA. The primary defect of the centromeric SMN2 copy gene is the production of the exon 7 skipped SMN protein. Since the "inclusion" of this exon has been shown to be perhaps the most critical molecular determinant in SMA pathogenesis, the focus of this proposal will be on the determinants that govern exon 7 splicing. The goals of Aims 1 and 2 are to identify the cis-elements that regulate SMN exon 7 RNA-processing, including exonic splice enhancers, silencers, flanking intronic elements, and the critical C/T transition within exon 7 (SMN1=C; SMN2=T). This will be accomplished by extensive genetic analysis and complemented by in vitro analysis of splicing. Extending the genetic work developed in the first two aims, Aim 3 will identify and characterize the trans factors that mediate proper processing of SMN exon 7 pre-mRNA. It will be determined whether factor binding to exon 7 correlates with the ability to stimulate exon 7 inclusion. These studies may be useful for the development of therapeutics that promote the inclusion of exon 7 in SMN2-derived mRNAs.

描述（由申请人提供）： 脊柱肌肉萎缩（SMA）是由于两个端粒5q13的损失而引起的 SMN1基因的副本。 centromeric 5q13 SMN2基因编码相同的 蛋白质。虽然SMN2的损失并没有导致SMA的发展，但 SMN2的存在以剂量依赖性的方式起作用 缺乏SMN1。 SMN1基因产生全长转录本，而 SMN2的主要产物是缺少外显子7的外显子兼的mRNA。我们报道 SMN1和SMN2之间的单个非晶核苷酸的差异为 此替代剪接的基础。通过比较衍生的混合SMN基因 从没有影响亲戚的SMA患者中，我们证明了 该核苷酸（SMN1或SMN2）决定了临床结果。因此， SMA的临床演变与SMN基因的剪接相关。 SMN 蛋白质自求解，蛋白质从替代品转换 剪接的SMN2 RNA，形成低聚物的能力降低，并且稳定较低 与全长蛋白相比。此外，来自SMA的SMN点突变体 患者表现出与疾病严重程度成正比的寡聚性缺陷。 这些研究定义了开发的遗传和生化基础 SMA。 centromeric SMN2副本基因的主要缺陷是产生 外显子7跳过SMN蛋白。由于已显示出该外显子的“包含” 为了成为SMA发病机理中最关键的分子决定因素， 该提案的重点将放在支配外显子7拼接的决定因素上。 目标1和2的目标是确定调节SMN的顺式元素 外显子7 RNA处理，包括外部剪接增强剂，消音器，侧翼 内含子元素以及外显子7中的临界C/T转变（SMN1 = C; smn2 = t）。这将通过广泛的遗传分析和 通过对剪接的体外分析进行补充。扩展遗传工作 在前两个目标中开发的AIM 3将识别和表征trans 介导SMN外显子7前MRNA的适当处理的因素。这将是 确定因子与外显子7的结合是否与能力相关 刺激外显子7包含。这些研究可能对发展 促进在SMN2衍生的mRNA中促进外显子7的治疗剂。