RESUMO
Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.
Assuntos
Mutação/genética , Splicing de RNA/genética , Retina/patologia , Descolamento Retiniano/genética , Epitélio Pigmentado da Retina/patologia , Simportadores de Sódio-Bicarbonato/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Descolamento Retiniano/patologia , Tomografia de Coerência Óptica/métodosRESUMO
Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.
Assuntos
Fases de Leitura Aberta/genética , Oryza/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Análise de Sequência de DNA/métodos , Biologia Computacional/métodos , DNA de Plantas/genética , Bases de Dados Genéticas , Genoma de Planta/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which â¼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution.
