Celf1 mediates post-transcription control of gene expression in lens development

Abstract

Purpose: While the significance of signaling and transcriptional control mechanisms is well appreciated in lens development, that of post-transcriptional regulation remains unexplored. To address this deficit, we applied the lens gene discovery tool iSyTE to identify the RNA binding protein (RBP) Celf (Cugbp1) and found that its deletion in mouse or knockdown in fish disrupts lens development and causes cataract. Here, we report on elucidation of the novel mechanistic basis of Celf1-mediated control of important regulators in lens development.

Methods: Immunostaining and qRT-PCR were used to test the expression of targets in Celf1 conditional knockout (cKO) mouse mutant lens. RNA immunoprecipitation (RIP) with Celf1 antibody tested the direct interaction of Celf1 with RNA targets. Celf1-mediated translational control was tested by cloning the 5’UTR of p27 mRNA into reporter plasmid upstream of firefly luciferase. Transient transfections introduced reporter vector into control or Celf1-knockdown (KD) mouse lens epithelial cell line 21EM15.

Results: While in normal E16.5 lens p27 protein is downregulated in centrally located fiber cells, Celf1 cKO mutant lens exhibits ectopic and up-regulated presence of this protein in this region. RIP analysis indicates that Celf1 directly binds to p27 mRNA and functional analysis demonstrates elevated luciferase expression in Celf1-KD cells, indicating that Celf1 potentially directly inhibits p27 translation in differentiating lens fiber cells. Interestingly, Prox1 protein is significantly up-regulated in Celf1 cKO lens epithelium, while other mRNAs are either up-regulated (p21) or down-regulated (Dnase2b) in Celf1 cKO lens. These data indicate that Celf1 functions to fine-tune gene expression by controlling translation or mRNA stability in both lens epithelial and fiber cells.

Conclusions: In past, based on the fiber cell-specific expression pattern of the RBP Tdrd7, we had anticipated a relatively straightforward model that different epithelium-specific or fiber-specific RBPs may function to control post-transcriptional gene expression in these cells. However, these new data demonstrate that a single RBP Celf1 expressed in both the epithelium and fiber cells can control their respective proteomes by distinct post-transcriptional mechanisms such as RNA stability or translational control, thus highlighting a previously unanticipated mechanistic sophistication in lens gene regulation.