TARGETING ABERRANT RNA METABOLISM IN CRX-LINKED RETINOPATHIES



 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.