TARGETING ABERRANT RNA METABOLISM IN CRX-LINKED RETINOPATHIES

 描述(申请人提供)：锥杆同源框转录因子CRX调节许多光感受器基因的表达，是光感受器发育和生存所必需的。人类CRX基因突变主要与常染色体显性遗传性视网膜病变有关：Leber先天性黑色素沉着症(AdLCA)、视锥细胞视杆细胞营养不良症(AdCRD)和视网膜色素变性(Adrp)，发病年龄和严重程度各不相同。目前对这些疾病的了解很少，也没有可用的治疗方法。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，即产生C末端截短CRX蛋白的移码/无义突变。这些突变蛋白保留了DNA结合，但缺乏转录调节活性，因此干扰了野生型(WT)蛋白的功能。III类动物模型包括CRX-E168d2和CRX-Tvrm65小鼠以及CRX-Rdy猫，它们都表现出类似于人类adLCA或adCRD的表型。这些模型揭示了一个意想不到的原发致病缺陷，即相对于WT的突变mRNA/蛋白的过度生产。这些过度突变的CRX产物“放大”了突变蛋白的毒性效应，它们的水平与表型严重程度直接相关。然而，目前还不清楚CRX突变是如何导致他们自己的等位基因选择性过度表达的。我们在这里提出的研究就是为了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mRNAs更稳定，这表明突变的CRX的过量生产主要是由于其转录的稳定性增加所致。我们还发现，CRX不仅可以与DNA结合，还可以作为RNA结合蛋白(RBP)与自己的转录本和视紫红质mRNA结合。这些发现使我们假设CRX在调节mRNA稳定性方面扮演了一个新的角色，特别是针对它自己的转录本。此外，III类CRX突变的存在改变了突变等位基因转录本的稳定性，导致突变的mRNA/蛋白质过度生产和随后的光感受器营养不良。为了验证这些假设，我们设计了一系列实验，以定位WT和突变的CRX mRNA中包含“稳定密码”的RNA序列，并确定这些密码如何被包括CRX在内的RNA结合蛋白解释；描述受CRX依赖的mRNA稳定性调节的其他光感受器基因；以及确定CRX突变对培养细胞和小鼠模型中这种调节的影响。这项研究将极大地促进我们对CRX的多功能作用和作用机制以及致病突变的影响的理解。它还可能对其他神经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.