Dataset Information

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

ABSTRACT: Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein compositions between the lens epithelium and fibers, we employed tandem mass spectrometry (2D-LC/MS) analysis of microdissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular processes and subcellular localizations, were adapted for the lens. Expression levels of both epithelial and fiber proteomes were compared with whole lens proteome and mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins (e.g. Carhsp1), translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal [e.g. non-muscle myosin IIA heavy chain (Myh9) and ?B2-spectrin (Sptbn2)] and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown functions in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets represent the first mouse lens epithelium and fiber cell proteomes, establish comparative analyses of protein and RNA-Seq data, and characterize the major proteome remodeling required to form the mature lens fiber cells.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC6360118 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

Zhao Yilin Y Wilmarth Phillip A PA Cheng Catherine C Limi Saima S Fowler Velia M VM Zheng Deyou D David Larry L LL Cvekl Ales A

Experimental eye research 20181022

Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development a ...[more]

PMID: 30359574

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Project description:Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular composition of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein composition between the lens epithelium and fibers, we employed tandem mass spectrometry (2DLC/ MS) analysis of micro-dissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular process and subcellular localization, were adapted for lens. Expression levels of both epithelial and fiber proteomes were compared with their temporal and spatial mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins, translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown function in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets established reference mouse lens epithelium and fiber cell proteomes, provided quantitative analyses of protein and RNA-Seq data, and probed the major proteome remodeling required to form the mature lens fiber cells.

Project description:The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions for three days and euthanized under red light (<1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥ ±2.0). Of the protein coding transcripts, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation.

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Dataset Information

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

Publications

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

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