Regulation of Retinal Development by RNA molecules

PROJECT SUMMARY The prevailing hypothesis in biology is encapsulated by the ‘central dogma’, which posits that our heritable genetic traits are encoded by DNA, transcribed into an RNA intermediate, and translated into functional proteins. However, this model fails to incorporate the contributions of the prevalent expression of non-coding RNA transcripts. Our recent profiling of the developing retina of humans and mice highlighted widespread and temporally dynamic expression of long non-protein coding RNAs (lncRNAs). lncRNAs play essential roles in the regulation of transcriptional and epigenetic programs during cell fate decisions, including those of the developing retina. One mechanism by which lncRNAs function is through initiating scaffolding complexes with proteins and DNA, including interactions with transcription factors to modulate transcriptional activity. We hypothesize that retinal expressed lncRNAs function to control retinal gene regulatory networks, in part through interaction with transcription factors. To test this hypothesis, we first propose to utilize both gain-of-function and a novel loss-of-function models to examine the effect of the lncRNA Gm11454 expression on retinal progenitor cell development and differentiation. Additionally, we will assess the trans functions of Gm11454 in virally mediated rescue experiments. Secondly, the sites of DNA-RNA interaction are determined through Chromatin Isolation by RNA Precipitation. The functional significance of sites of DNA-RNA binding will be determined by assessing changes gene expression and chromatin accessibility because of altered Gm11454 expression. Thirdly, we will characterize RNA-transcription factor interactions. Preliminary results identified Gm11454- transcription factor interactions. We will test the significance of RNA-binding by transcription factors through genome-wide profiling of RNA-guided transcription factor binding to DNA. Finally, we will implement a novel Digital Affinity Profiling via Proximity Ligation approach to identify novel RNA-interactions of retinal transcription factors, performing extensive validation techniques. These studies will provide important insights into the biomolecular regulatory mechanisms by which RNA transcripts control retinal gene regulatory networks. Importantly, these studies aim to provide novel insights into the molecular consequences of altered (lnc)RNA expression during retinal development and disease.