Abstract：
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-/- 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.
摘要：
晶状体纤维细胞的分化涉及来自生长因子的信号的复杂相互作用，以及通过转录和转录后调节因子严格调节的基因表达。各种研究表明，RNA结合蛋白，在核糖核蛋白颗粒中发挥作用，在晶状体发育过程中调节转录后表达具有重要作用。在这项研究中,我们检查了BTG/TOB家族RNA结合蛋白的两个成员的表达和定位，TOB1和TOB2，在发育中的晶状体中，检测了缺少Tob1的小鼠的表型。通过RT-PCR,在胚胎和出生后小鼠晶状体的上皮细胞和纤维细胞中均检测到Tob1和Tob2mRNA。原位杂交显示Tob1和Tob2mRNA在早期分化纤维中表达最强烈，在前上皮细胞中表达较弱，两者似乎都在E15.5镜片的发芽区下调。从E11.5到E16.5检测到TOB1蛋白，主要在早期分化的成纤维细胞中的大型细胞质点中检测到。通常与P体标记共定位，DCP2.还观察到偶尔的核点。相比之下,在一系列相互连接的核周颗粒中检测到TOB2，在后来分化内部皮质的成纤维细胞中。TOB2似乎未与DCP2共定位，但与早期应激颗粒标记（EIF3B）部分共定位。这些数据表明TOB1和TOB2涉及晶状体纤维细胞中mRNA加工周期的不同方面。使用或不使用纤维分化剂量的FGF2处理的大鼠晶状体上皮外植体的体外实验表明，在FGF诱导的分化过程中，TOB1和TOB2均上调。在区分外植体时，TOB1也与DCP2共定位在大的细胞质颗粒中。对Tob1-/-小鼠的分析显示晶状体形态相对正常，但在赤道和E13.5胚胎的晶状体纤维团中某些细胞的细胞周期停滞存在细微缺陷。总的来说,这些发现表明，TOB蛋白在晶状体纤维分化过程中的RNA加工中起着不同的调节作用。
