TARGETING ABERRANT RNA METABOLISM IN CRX-LINKED RETINOPATHIES

 描述（由申请人提供）：视锥-视杆同源框转录因子CRX调节许多光感受器基因的表达，并且是光感受器发育和存活所必需的。人类CRX突变主要与常染色体显性（ad）视网膜病相关：Leber先天性黑蒙（adLCA）、视锥-视杆细胞营养不良（adCRD）和色素性视网膜炎（adRP），发病年龄和严重程度各不相同。这些疾病目前知之甚少，没有治疗方法。最近的体外和动物模型研究揭示了三种不同类型的CRX突变的病理机制。我们的实验室已经广泛研究了III类突变，移码/无义突变产生的C-末端截短CRX蛋白。这些突变蛋白保留DNA结合，但缺乏转录调节活性，因此干扰野生型（WT）蛋白功能。III类的动物模型包括Crx-E168 d2和Crx-Tvrm 65小鼠和Crx-Rdy猫，所有动物均显示出与人类adLCA或adCRD相似的表型。这些模型揭示了一个意想不到的主要致病缺陷，相对于WT的突变mRNA/蛋白质的过度生产。这些过量的突变Crx产物“放大”突变蛋白的毒性作用，并且它们的水平与表型严重性直接相关。然而，目前还不清楚Crx突变如何导致其自身等位基因的选择性过表达。我们在这里提出的研究旨在在分子水平上解决这个问题。我们的初步结果表明，正常的Crx mRNA是短暂的，但携带III类突变的mRNA要稳定得多，这表明突变型CRX的过度产生主要是由其转录本的稳定性增加引起的。我们还发现CRX不仅与DNA结合，而且还作为RNA结合蛋白（RBP）与其自身的转录物和视紫红质mRNA结合。这些发现使我们假设CRX在调节mRNA稳定性方面的新作用，特别是针对其自身的转录物。此外，III类CRX突变的存在改变了突变等位基因转录物的稳定性，导致突变mRNA/蛋白质过度产生和随后的光感受器营养不良。为了验证这些假设，我们设计了一组实验来绘制WT和突变型Crx mRNA中含有“稳定性密码”的RNA序列，并确定这些密码如何被RNA结合蛋白（包括CRX）解释;分析其他受CRX依赖性mRNA稳定性调控的感光基因;并确定Crx突变对培养细胞和小鼠模型中这种调控的影响。这项研究将大大促进我们对CRX的多功能作用和作用机制以及致病突变的影响的理解。它也可能对其他神经系统疾病产生更广泛的影响。

 DESCRIPTION (provided by applicant): The cone-rod homeobox transcription factor CRX regulates expression of many photoreceptor genes and is required for photoreceptor development and survival. Human CRX mutations are mostly associated with autosomal dominant (ad) retinopathies: Leber congenital amaurosis (adLCA), cone-rod dystrophy (adCRD) and retinitis pigmentosa (adRP), with variable age of onset and severity. These diseases are currently poorly understood and no treatments are available. Recent in vitro and animal model studies have shed light on the pathologic mechanisms underlying three distinct classes of CRX mutations. Our laboratory has extensively studied Class III mutations, frameshift/nonsense mutations producing C-terminal truncated CRX proteins. These mutant proteins retain DNA binding but lack transcriptional regulatory activity and so interfere with wild- type (WT) protein function. Animal models for Class III include Crx-E168d2 and Crx-Tvrm65 mice and Crx-Rdy cats, all showing similar phenotypes that resemble adLCA or adCRD in humans. These models have revealed an unexpected primary pathogenic defect, overproduction of the mutant mRNA/protein relative to WT. These excessive mutant Crx products "amplify" the toxic effect of the mutant protein and their level directly correlates with phenotype severity. However, it is unclear how Crx mutations cause this selective overexpression of their own allele. The study we propose here is designed to address this question at the molecular level. Our preliminary results suggest that the normal Crx mRNA is short-lived, but mRNAs carrying Class III mutations are much more stable, indicating that the overproduction of mutant CRX is primarily caused by the increased stability of its transcript. We also discovered that CRX not only binds to DNA, but also acts as a RNA-binding protein (RBP) to bind to both its own transcript and Rhodopsin mRNA. These findings lead us to hypothesize a new role for CRX in regulating mRNA stability, particularly targeting its own transcript. Furthermore, the presence of Class III CRX mutations alters the stability of the mutant allele's transcripts, resulting in mutant mRNA/protein overproduction and subsequent photoreceptor dystrophy. To test these hypotheses, we have designed a set of experiments to map RNA sequences containing the "stability codes" in WT and mutant Crx mRNA, and to determine how these codes are interpreted by RNA binding proteins, including CRX; to profile other photoreceptor genes subject to CRX-dependent mRNA stability regulation; and to determine the effects of Crx mutations on this regulation in both cultured cells and mouse models. This study will significantly advance our understanding of CRX's multifunctional roles and mechanisms of action, and the effects of disease-causing mutations. It could also have much broader implications for other neurological disorders.