Micro-RNAs direct a ubiquitous mode of post-transcriptional gene regulation in plants and animals which has been implicated in a vast number of cellular processes and disease pathologies. Within the visual system, micro-RNAs are essential for proper development and survival of retinal neurons, particularly after stress. A deeper understanding of the physiological impact of micro-RNAs has, in the past, been limited by lack of a robust, systematic method for studying the interaction of micro-RNAs with their target transcripts. The recently developed technology of CrossLinking ImmunoPrecipitation followed by deep sequencing (CLIPseq.) has provided an elegant solution to this problem. It is now possible to crosslink the major protein component of the micro-RNA induced silencing complex to target transcripts in vivo using ultraviolet light. These covalently linked protein-RNA complexes can then be purified by high stringency immunoprecipitation and the RNA fragments sequenced using massively parallel sequencing technology. This unbiased, biochemical approach facilitates the creation of a genome-wide map of micro-RNA regulation. CLIP-seq. technology, along with the power of mouse genetics, will be used to identify the gene targets of this regulation, and to assess the impact of micro-RNA gene regulation on the health and physiology of rod photoreceptors. First, genetically modified mice lacking most mature micro-RNAs specifically in rod photoreceptors will be generated. The impact of this modification on the anatomy and physiology of the retina will be assessed. Finally, three complementary approaches will be employed to systematically identify the targets of micro-RNA gene regulation in rod photoreceptors. Integration of the results from these three approaches will lead to a high confidence map of genes targeted by micro-RNAs in rods. As micro-RNAs are essential to the survival of rods, particularly under stress, the proposed experiments will bring to light new insight into pathways that mediate survival of photoreceptors under cellular stress. Such insight promises to identify new targets and approaches for the treatment of blinding diseases. In addition, these projects will provide a unique training experience, allowing the candidate the opportunity to master both classical techniques in visual system biology and mouse genetics along with emerging high resolution imaging technologies and next generation sequencing analysis. When combined with the candidate's previously earned expertise in RNA molecular biology, this new repertoire of techniques will prepare the candidate for a promising career independently investigating post-transcriptional gene regulation in the visual system.
Photoreceptor cell death is the primary cause of blindness in visual system diseases such as retinitis pigmentosa, Stargardt disease, and age-related macular degeneration. Therefore, dissecting the molecular events underlying protection of photoreceptors from stress-induced cell death is of paramount importance to understanding the pathology of these diseases and developing interventions to treat or prevent them. The studies described here promise to provide much insight into mechanisms for promoting photoreceptor survival, and maintenance of vision.
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|Sundermeier, Thomas R; Vinberg, Frans; Mustafi, Debarshi et al. (2014) R9AP overexpression alters phototransduction kinetics in iCre75 mice. Invest Ophthalmol Vis Sci 55:1339-47|