Structure of the dystrophin rod in relation to exon boundaries and exon skipping

描述(由申请人提供)：本项目的目的是了解肌营养不良蛋白的杆状区域的性质。虽然这个区域构成了dystrophin的大部分序列，但由于中间的dystrophin与其他细胞成分的相互作用，更小的末端球状结构域通常被认为更重要。在许多情况下，Dystrophin的缩短版本(其中杆状区域的一部分被移除)-所谓的迷你Dstrophin-正在作为可能的基因治疗替代品进行研究。然而，在动物模型中已经看到了实质性的上下文效应，在这些动物模型中，结构略有不同的迷你营养不良蛋白涉及不同的杆状修饰，具有明显不同的效果。此外，最近已经证明，dystrophin基因的处理通过跳过外显子自然产生各种缺失(至少在RNA水平上)，这可能是治疗上的利用。然而，营养不良蛋白棒的生物物理和生化结构还不是很清楚。它传统上被认为是由一些重复的模体和一些散布的所谓铰链区域组成的。然而，我已经证明了这些区域相互作用的方式是不同的--一些基序似乎独立于它们的邻居，而另一些则不是，表现出强烈的合作结构相互作用。这对编辑有明显的影响，因为在合作区域进行编辑可能会产生远端影响。同样，正常的RNA剪接事件似乎通过外显子跳过产生自然的缺失变体，但这种模式令人费解，因为大多数编辑并不发生在这些重复基序的连接处(几乎只在人工删除中使用)，而是在中途发生，从而产生新的混合基序。所有这些因素都导致了产生功能性编辑的抗肌营养不良蛋白变体的不可预测性。我们试图制作dystrophin杆的结构域结构的生物物理和生化图，从而为编辑该分子提供合理的基础，并更好地理解由正常的差异RNA加工产生的可能的变体。 这项工作旨在了解dystrophin(Duchenne肌营养不良症中存在缺陷的蛋白质)的棒状区的结构，它是该蛋白质的最大组成部分，也是导致DMD的大多数突变发生的地方。这种棒在蛋白质和DNA水平上都由许多重复区域组成，它的组装方式表明它可以被编辑来改善这些缺陷。在许多正在开发的治疗策略中，我们设想了这种棒的编辑和缩短版本；我们将确定如何在保持蛋白质整体稳定的情况下产生这些缩短的区域。

DESCRIPTION (provided by applicant): The aim of this project is to understand the nature of the rod region of dystrophin. Although this region makes up the bulk of dystrophin's sequence, the much smaller terminal globular domains are often considered more important since the mediate dystrophin's interactions with other cellular components. In many cases, shortened versions of dystrophin in which part of the rod region is removed - the so-called mini- dystrophins - are being studied as possible gene therapy replacements. However, substantial context effects have been seen in animal models, in which slightly differently constructed mini-dystrophins involving different rod modifications have had markedly different efficacies. As well, it has recently been demonstrated that the processing of the dystrophin gene naturally produces various deletions (at least at the RNA level) though exon skipping, which may be therapeutically harnessed. However, the biophysical and biochemical construction of the dystrophin rod is not well understood. It is traditionally thought of as composed of a number of repetitive motifs with a few interspersed so-called hinge regions. However, I have shown that the manner in which these regions interact is heterogeneous - some motifs appear independent of their neighbors, while others are not, exhibiting strongly cooperative structural interactions. This has obvious implications for editing, since editing in a cooperative region can have distal effects. As well, normal RNA splicing events appear to produce natural deletion variants via exon skipping, but the pattern is perplexing, in that most edits do not occur near the junctions of these repeat motifs (as have been nearly exclusively employed in man-made deletions), but midway through, resulting in novel, hybrid motifs. All of these factors contribute to the unpredictability of producing functional edited dystrophin variants. We seek to produce a biophysical and biochemical map of the domain structure of the dystrophin rod, thereby providing a rational basis to edit this molecule, and a better understanding of the putative variants produced by normal differential RNA processing. This work seeks to understand the structure of the rod region of dystrophin (the protein defective in Duchenne Muscular Dystrophy), which is the largest component of this protein, and is where most mutations responsible for DMD occur. This rod is composed of many repetitive regions at both the protein and DNA levels, and the manner in which it is assembled suggests that it can be edited to ameliorate these defects. In many therapeutic strategies under development, edited and shortened versions of this rod are envisioned; and we will determine how to produces these shorted regions while still maintaining stability of the protein as a whole.