TOB1 and TOB2 mark distinct RNA processing granules in differentiating lens fiber cells.

Differentiation of lens fiber cells involves a complex interplay of signals from growth factors together with tightly regulated gene expression via transcriptional and post-transcriptional regulators. Various studies have demonstrated that RNA-binding proteins, functioning in ribonucleoprotein granules, have important roles in regulating post-transcriptional expression during lens development. In this study, we examined the expression and localization of two members of the BTG/TOB family of RNA-binding proteins, TOB1 and TOB2, in the developing lens and examined the phenotype of mice that lack Tob1. By RT-PCR, both Tob1 and Tob2 mRNA were detected in epithelial and fiber cells of embryonic and postnatal murine lenses. In situ hybridization showed Tob1 and Tob2 mRNA were most intensely expressed in the early differentiating fibers, with weaker expression in anterior epithelial cells, and both appeared to be downregulated in the germinative zone of E15.5 lenses. TOB1 protein was detected from E11.5 to E16.5 and was predominantly detected in large cytoplasmic puncta in early differentiating fiber cells, often co-localizing with the P-body marker, DCP2. Occasional nuclear puncta were also observed. By contrast, TOB2 was detected in a series of interconnected peri-nuclear granules, in later differentiating fiber cells of the inner cortex. TOB2 did not appear to co-localize with DCP2 but did partially co-localize with an early stress granule marker (EIF3B). These data suggest that TOB1 and TOB2 are involved with different aspects of the mRNA processing cycle in lens fiber cells. In vitro experiments using rat lens epithelial explants treated with or without a fiber differentiating dose of FGF2 showed that both TOB1 and TOB2 were up-regulated during FGF-induced differentiation. In differentiating explants, TOB1 also co-localized with DCP2 in large cytoplasmic granules. Analyses of Tob1