Ribosomal targeting strategy and nuclear labeling to analyze photoreceptor phosphoinositide signatures

Abstract

Reversible phosphorylation of phosphatidylinositol by phosphoinositide (PI) kinases and phosphatases generates seven distinct phosphoinositide phosphates, called phosphoinositides or PIPs. All seven PIPs are formed in the retina and photoreceptor cells. Around 50 genes in the mammalian genome encode PI kinases and PI phosphatases. There are no studies available on the distribution of these enzymes in the retina and photoreceptors. AimTo employ Ribosomal Targeting Strategy and Nuclear Labeling to Analyze Phosphoinositide Signatures in rod-photoreceptor cells. MethodsHA-tagging of ribosomal protein Rpl22 was induced with Cre-recombinase under the control of the rhodopsin promoter. Actively translating mRNAs associated with polyribosomes were isolated by immunoprecipitation with HA antibody, followed by RNA isolation and gene identification. We also isolated biotinylated-rod nuclei from NuTRAP mice under the control of the rhodopsin-Cre promoter and analyzed nuclear phosphoinositides. ResultsOur results indicate that the expression of class I and class III PI 3-kinase, PI4K IIIβ, PI 5-kinase, PIKfyve, PI3-phosphatases, MTMR2, 4, 6, 7, 14, PI4-phosphatase, TMEM55A, PI 5-phosphatases, SYNJI, INPP5B, INPP5E, INPP5F, SKIP and other phosphatases with dual substrate specificity, PTPMT1, SCAM1, and FIG4 are highly enriched in rod photoreceptor cells compared with the retina and cone-like retina. Our analysis identified the presence of PI(4)P, PI(3,4)P2, PI(3,5)P2, and PI(4,5)P2 in the rod nuclei. ConclusionsOur studies for the first time demonstrate the expression of PI kinases, PI phosphatases, and nuclear PIPs in rod photoreceptor cells. The NuTRAP mice may be useful not only for epigenetic and transcriptomic studies but also for in vivo cell-specific lipidomics research.