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Nuclear retention is a mechanism whereby excess RNA transcripts are stored in the event that a cell needs to quickly respond to a stimulus; maintaining proper nuclear-to-cytoplasmic balance is important for cellular homeostasis and cell function. There are many mechanisms that are employed to determine whether to retain a transcript or export it to the cytoplasm, although the extent to which tissue or cell type, internal and external stressors, and disease pathogenesis affect this process is not yet clear. As the most biochemically active tissue in the body, the retina must mitigate endogenous and exogenous stressors to maintain cell health and tissue function. Oxidative stress, believed to contribute to the pathogenesis or progression of age-related macular degeneration (AMD) and inherited retinal dystrophies (IRDs), is produced both internally from biochemical processes as well as externally from environmental insult. Here, we evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), with specific focus on transcripts related to RPE function and disease.We performed poly(A) RNA sequencing on nuclear and cytoplasmic fractions from human induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide (Hsub2/subOsub2/sub), as well as on untreated controls.Under normal conditions, the number of mRNA transcripts retained in the nucleus exceeded that found in studies on other tissues. Further, the nuclear-to-cytoplasmic ratio of transcripts was altered following oxidative stress, as was the retention of genes associated with AMD and IRDs, as well as those that are important for RPE physiology.These results provide a localization catalog of all expressed mRNA in iPSC-RPE under normal conditions and after exposure to Hsub2/subOsub2/sub, shedding light on the extent to which Hsub2/subOsub2/subalters transcript localization and potentially offering insight into one mechanism through which oxidative stress may contribute to the progression of visual disorders.

Nuclear retention is a mechanism whereby RNA transcripts are held in the nucleus to maintain a proper nuclear-to-cytoplasmic balance or as a stockpile for use in responding to stimuli. Many mechanisms are employed to determine whether transcripts are retained or exported to the cytoplasm, though the extent to which tissue- or cell-type, stressors, or disease pathogenesis affect this process remains unclear. As the most biochemically active tissue in the body, the retina must mitigate endogenous and exogenous stressors to maintain cell health and tissue function. Oxidative stress, believed to contribute to the pathogenesis, or progression, of age-related macular degeneration (AMD) and inherited retinal dystrophies (IRDs), is produced both internally from biochemical processes, as well as externally from environmental insult. To evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), we performed poly-A RNA sequencing on nuclear and cytoplasmic fractions from induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide, as well as untreated controls. Under normal conditions, the number of mRNA transcripts retained in the nucleus exceeded that found in studies of other tissues. Further, the nuclear-to-cytoplasmic ratio of transcripts is altered following oxidative stress, as is the retention of genes associated with AMD, IRDs, and those important for RPE physiology. These results provide a retention catalog of all expressed mRNA in iPSC-RPE under normal conditions and after exposure to hydrogen peroxide, offering insight into one of the potential roles oxidative stress plays in the progression of visual disorders.

Both transcriptional and post-transcriptional regulation of gene expression play significant roles in diverse biological processes, but little is known about how post-transcriptional regulation impacts retinal development. Here we report our study of the function of two members of the TTP (tristetraprolin) mRNA binding protein family, Zfp36l1 and Zfp36l2, in the developing retina. TTP proteins are highly conserved CCCH zinc finger proteins, which carry out their functions by promoting target mRNA decay and modulating translation. We found that Zfp36l1 and Zfp36l2 were expressed in retinal progenitor cells (RPCs) during development and Müller glial cells and photoreceptors in the mature retina. Our analysis of the mutant retinas showed that, whereas the single knockout retinas were largely normal, the double knockout (DKO) retina showed decreased RPC proliferation and increased differentiation of multiple retinal cell types. RNA-seq analysis confirmed the imbalance of proliferation and differentiation in the DKO retina. Gene ontology and in silico target gene analysis indicates that Zfp36l1 and Zfp36l2 exert their function by directly regulating multiple classes of proteins, including components of multiple signaling pathways such as the sonic hedgehog pathway and the Notch pathway, cell cycle regulators, and most interestingly transcription factors directly involved in retinal differentiation. These results reveal a new tier of gene regulation controlling retinal development.

In the current scientific era, where the next-generation sequencing is a commonplace, we are just now beginning to understand the important role of the noncoding genome in diseases of the human eye. Of the noncoding genes, microRNAs (miRNAs) and the less understood, long noncoding RNAs (lncRNAs) have recently become the focus of an intense research effort, and a growing body of evidence implicates these RNAs and their dysregulation in various disease processes. Here, we examine the connections between these noncoding elements and several eye diseases, including age-related macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa, and retinoblastoma.

RNA-binding proteins (RBPs) are instrumental in the biochemical processing and physiological functioning of non-coding RNAs. Therefore, as interest in non-coding RNAs continues to expand, refining the techniques capable of probing protein-RNA interactions will prove ever more valuable in the characterization of these molecules. To identify the RNAs bound by a given RBP, cross-linking and immunoprecipitation (CLIP) and its iterations have been widely utilized, but these approaches can be complex, labor-intensive, and time consuming. Here, we describe a rapid and technically simple method based upon individual nucleotide resolution CLIP (iCLIP) and infrared CLIP (irCLIP). Termed quick-irCLIP, our protocol circumvents confounding steps, can be completed in less than three days, and is capable of interrogating protein-RNA interactions at single nucleotide resolution. Protocol name: Quick-irCLIP – rapid infrared adaptor based individual nucleotide resolution UV cross-linking and immunoprecipitation, Keywords: quick-irCLIP, iCLIP, irCLIP, CLIP, RNA-binding protein, RNA, Protein-RNA interaction

As with other retinal cell types, retinal ganglion cells (RGCs) arise from multipotent retinal progenitor cells (RPCs), and their formation is regulated by a hierarchical gene-regulatory network (GRN). Within this GRN, three transcription factors--atonal homolog 7 (Atoh7), POU domain, class 4, transcription factor 2 (Pou4f2), and insulin gene enhancer protein 1 (Isl1)--occupy key node positions at two different stages of RGC development. Atoh7 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Pou4f2 and Isl1 are downstream and regulate RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key step in RGC development, remains unclear. Here we report that ectopic expression of Pou4f2 and Isl1 in the Atoh7-null retina using a binary knockin-transgenic system is sufficient for the specification of the RGC fate. The RGCs thus formed are largely normal in gene expression, survive to postnatal stages, and are physiologically functional. Our results indicate that Pou4f2 and Isl1 compose a minimally sufficient regulatory core for the RGC fate. We further conclude that during development a core group of limited transcription factors, including Pou4f2 and Isl1, function downstream of Atoh7 to determine the RGC fate and initiate RGC differentiation.