RNA-binding proteins in the mouse lens: Functional classifications, expression profiling, and interaction studies of Carhsp1 with crystallin mRNAs

RNA-binding proteins (RBPs) are critical regulators of mRNAs controlling all processes such as RNA transcription, transport, localization, translation, mRNA:ncRNA interactions, and decay. Cellular differentiation is driven by temporally and spatially regulated expression of proteins needed for the optimal function of individual cells, tissues and organs. Lens fiber cell differentiation is marked by high levels of expression of crystallin genes encoding critical proteins for lens transparency and light refraction. Herein we performed proteomic and transcriptomic analyses of RBPs in differentiating mouse lenses to identify the most abundant RBPs and establish dynamic changes of their expression in differentiating lenses. Expression analyses showed highly abundant RBPs, including Carhsp1, Igf2bp1/ZBP1, Ybx1, Pabpc1, Ddx39, and Rbm38. Binding sites of Carhsp1, the most abundant lens RBP, were predicted in various crystallin and β-actin mRNAs. Immunoprecipitations using Carhsp1-specific antibodies confirmed interactions of Carhsp1 with crystallin mRNAs in newborn lens. A combination of single molecule RNA FISH (smFISH) and immunofluorescence was used to probe in vivo interactions of Carhsp1 with αA-, αB-crystallin, and β-actin mRNAs in cytoplasm and nucleoplasm of cultured mouse lens epithelial cells. These experiments favor a working model of direct association of Carhsp1 mediated by multiple candidate binding sites within both αA-, αB-crystallin mRNAs. Together, these results open new avenues to perform comprehensive genetic, cell, and molecular biology studies of individual RBPs in the lens.