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Sharda Yadav, Hong Hao, Marie-Audrey Kautzmann, Matthew Brooks, Jacob Nellissery, Anand Swaroop; Transcription and splicing associated protein NonO/p54nrb regulates rod specific genes including rhodopsin and their regulators required for rod differentiation and homeostasis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2614.
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© ARVO (1962-2015); The Authors (2016-present)
We have identified Non pou domain containing octamer binding protein (NonO) from bovine retinal extracts using the rhodopsin distal enhancer region. The purpose of current study is to elucidate the role of NonO in regulating the expression of rhodopsin.
To understand role of NonO in regulation of rhodopsin expression we performed shRNA knockdown of NonO using in vivo electroporation. P0 CD1 pups were co-electroporated with Rhodopsin promoter driving DsRed reporter along with either control or NonO shRNA. To visualize the electroporated cells, both shRNA constructs contained GFP reporter driven by signal recognition particle alpha promoter. GFP and DsRed positive cells in outer nuclear layer of the control and NonO shRNA electroporated retinas were examined. To explore in vivo role of NonO in rod photoreceptors of P21 retinas, chromatin immunoprecipitation was performed followed by deep sequencing (Chip-Seq). We choose zeitgeber (ZT) 6 and ZT18 as genes implicated in rod outer segment disc renewal exhibit differential expression at these times. Occupancy of NonO on gene promoters was analyzed using Genomatix software and UCSC genome browser.
Knockdown of NonO by shRNA resulted in death of developing rod photoreceptors. The rod death could be partially rescued by a truncated NonO, which contained C-terminal 224 to 473 amino acids. Luciferase reporter assays showed that C-terminal region of NonO is sufficient to activate Rhodopsin promoter luciferase reporter. Chip-Seq data suggested that NonO occupied genes are mostly involved in phototransduction pathway. NonO occupancy on the phototransduction genes is higher at ZT6 compared to ZT18. These results suggest that NonO is involved in circadian regulation of rhodopsin as well as other phototransduction genes and their regulators in mouse rod photoreceptors.
NonO is critical for rod development and its knockdown leads to rod cell death. C-terminal 224 to 473 amino acids of NonO are sufficient to activate rhodopsin promoter-luciferase reporter expression in vitro. Chip-Seq results suggest that NonO plays an important role in modulating the levels of phototransduction genes during the circadian cycle. Further analysis of NonO function in mice retina should provide new insights into the regulation of rod outer segment biogenesis.
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