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

描述（由申请人提供）：本项目旨在了解肌营养不良蛋白棒状区域的性质。虽然这个区域构成了肌营养不良蛋白序列的大部分，但由于介导肌营养不良蛋白与其他细胞成分的相互作用，通常认为更小的末端球状结构域更为重要。在许多情况下，缩短版本的肌营养不良蛋白，其中部分杆状区域被移除，即所谓的迷你肌营养不良蛋白，正在研究作为可能的基因治疗替代品。然而，在动物模型中已经看到了实质性的环境效应，在动物模型中，涉及不同杆修饰的结构略有不同的微型肌营养不良蛋白具有明显不同的功效。此外，最近有研究表明，肌营养不良蛋白基因的加工通过外显子跳变自然地产生各种缺失（至少在RNA水平上），这可能在治疗上加以利用。然而，肌营养不良蛋白棒的生物物理和生化结构尚不清楚。传统上认为它是由许多重复的图案和一些散布的所谓铰链区域组成的。然而，我已经表明，这些区域相互作用的方式是异质的——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以产生远端影响。同样，正常的RNA剪接事件似乎通过外显子跳跃产生自然的缺失变异，但这种模式令人困惑，因为大多数编辑不会发生在这些重复基序的连接处附近（几乎完全用于人造缺失），而是发生在中间，导致新的杂交基序。所有这些因素都导致产生功能性编辑的肌营养不良蛋白变体的不可预测性。我们试图绘制出肌营养不良蛋白棒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。

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.