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

描述（由申请人提供）：该项目的目的是了解肌营养不良蛋白杆区的性质。尽管该区域构成了肌营养不良蛋白序列的大部分，但较小的末端球状结构域通常被认为更重要，因为介导肌营养不良蛋白与其他细胞成分的相互作用。在许多情况下，正在研究缩短型抗肌营养不良蛋白，其中杆状区域的一部分被移除，即所谓的微型抗肌营养不良蛋白，作为可能的基因治疗替代品。然而，在动物模型中观察到了显着的背景效应，其中涉及不同杆修饰的稍微不同结构的微型肌营养不良蛋白具有显着不同的功效。此外，最近已经证明，抗肌营养不良蛋白基因的加工通过外显子跳跃自然产生各种缺失（至少在 RNA 水平），这可以在治疗上利用。然而，肌营养不良蛋白杆的生物物理和生化结构尚不清楚。传统上认为它由许多重复的图案和一些散布的所谓铰链区域组成。然而，我已经表明，这些区域相互作用的方式是异质的——一些图案看起来独立于它们的邻居，而另一些则不然，表现出强烈的合作性结构相互作用。这对编辑具有明显的影响，因为在合作区域中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跃产生自然缺失变体，但这种模式令人困惑，因为大多数编辑并不发生在这些重复基序的连接处附近（几乎专门用于人为删除），而是发生在中间，从而产生新的混合基序。所有这些因素都导致了功能性编辑抗肌萎缩蛋白变体产生的不可预测性。我们试图绘制抗肌萎缩蛋白杆结构域的生物物理和生化图谱，从而为编辑该分子提供合理的基础，并更好地理解正常差异RNA加工产生的假定变体。 这项工作旨在了解抗肌营养不良蛋白（杜氏肌营养不良症中存在缺陷的蛋白质）杆状区域的结构，该区域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。该杆在蛋白质和 DNA 水平上由许多重复区域组成，其组装方式表明可以对其进行编辑以改善这些缺陷。在许多正在开发的治疗策略中，都设想了该棒的编辑和缩短版本；我们将确定如何产生这些短路区域，同时仍保持整个蛋白质的稳定性。

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.