MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

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The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. This will help us gain a better understanding of the RPE in normal and disease states and provide us with tools for further analysis of the RPE. To this end, we have been studying the function and gene regulation of RPE65, a novel conserved developmentally regulated 65 kDa RPE-specific protein. We have cloned full-length human and mouse genes for RPE65. Comparison of the bovine-, human- and partial mouse-deduced protein sequences reveals a highly conserved molecule with about 99 percent similarity between species. We have made promoter/reporter constructs by using human 5' flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene and tested these in transient transfection assays in primary RPE cell cultures to identify transcriptional regulatory regions. We found that the human RPE65 gene 5' flanking region drives RPE-specific expression of the reporter gene. This is being further analyzed. The mouse 5' flanking region has also been sequenced and compared with the human, revealing conserved blocks, including identified transcriptional factor-binding elements in the putative promoter region. We have generated transgenic animals using mouse RPE65 gene promoter/reporter constructs for in vivo analysis of promoter function. These animals are currently being analyzed. To further explore the role of this gene in the RPE as well as possible effects on the neural retina, we are generating an RPE65 knockout mouse. The mouse genomic sequences have been used to construct a targeting vector for electroporation of mouse embryonic stem cells. In addition to regulation at the transcriptional level, RPE65 expression is also regulated post-transcriptionally at the level of translation. We have identified distinct sequences in the 3' untranslated region (UTR) of the RPE65 mRNA that control the stability and the efficiency of translation of the RPE65 message. Further analysis suggests that the effect may be at least partially coding-region sequence specific because the 3' UTR sequence affecting translational efficiency had no effect on translation of a foreign message. Possible implications of this are being studied.