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

Abstract

Abstract Lens fiber cell differentiation involves a complex interplay of growth factor signals and tight control of gene expression via transcriptional and post-transcriptional regulators. Recent studies have demonstrated an important role for RNA-binding proteins, functioning in ribonucleoprotein granules, in regulating post-transcriptional expression during lens development. Here we have documented the expression of TOB1 and TOB2, members of the BTG/TOB family of RNA-binding proteins, in the developing lens and examine the phenotype of mice that lack Tob1. Both Tob1 and Tob2 mRNA were detected by RT-PCR in epithelial and fiber cells of embryonic and postnatal lenses. By in situ hybridization, Tob1 and Tob2 mRNA were most intensely expressed in the early differentiating fibers, with weaker expression in anterior epithelial cells, and were down-regulated 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 co-localize with DCP2 but partially co-localized with a marker of stress granules (EIF3B). These data suggest that TOB1 and TOB2 are involved with different parts 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-/- mice revealed relatively normal lens morphology but a subtle defect in cell cycle arrest of some cells at the equator and in the lens fiber mass of E13.5 embryos. Overall, these findings suggest that TOB proteins play distinct regulatory roles in RNA processing during lens fiber differentiation.