The post-transcriptional regulators Celf1 and Elavl1 control key transcription factors in lens development

Abstract

Purpose : The mechanistic basis of the regulation of lens development is largely limited to our understanding of a few key lens transcription factors (TFs) and signaling molecules. Over the past few years, we have demonstrated that conserved RNA-binding proteins (RBPs) mediate post-transcriptional regulation of gene expression to control the cellular proteome of lens epithelial and fiber cells during development. These findings lead to important questions such as (1) do RBPs operate to control dosage of key TFs in the lens, and (2) do RBPs–similar to TFs–coordinately regulate gene expression in the lens? Here, we investigated the function of two RBPs, Celf1 and Elavl1, in the control of key TFs in lens development.

Methods : Compound conditional lens-specific Celf1 knockout mice (Celf1^cKO/lacZKI) were generated by crossing mice containing Pax6GFPCre, Celf1 floxed and germline knock-in alleles. Lens-specific Elavl1 conditional KO mice were generated by crossing Pax6GFPCre and Elavl1 floxed alleles. Immunofluorescence and immunoprecipitation (IP) were performed with specific antibodies. RNA-IP (RIP) and Cross-linked IP (CLIP) were performed on post-natal day (P) 15 and P13 lenses, respectively. Celf1 and Elavl1 stable knockdown (KD) cell lines were generated using shRNA.

Results : Lens epithelium and fiber cells of Cefl1^cKO/lacZKI animals showed abnormally high levels of Pax6 and Prox1 proteins. However, Cefl1^cKO/lacZKI isolated epithelium did not have significantly altered Pax6 and Prox1 mRNA, suggesting that Celf1-based regulation was mediated on the post-transcriptional level. This was further supported by the findings that Celf1-KD cells exhibited elevated Pax6 and Prox1 while Celf1 overexpression led to their reduction. Co-IP assays showed that Celf1 protein associates with Elavl1 protein in lens cells and RIP and CLIP assays showed that both Celf1 and Elavl1 proteins bind to Pax6 and Prox1 mRNAs in the lens, suggesting their coordinate control of these factors. Finally, Elavl1^cKO/cKO mice exhibit microphthalmia, further supporting its involvement in eye development.

Conclusions : This work shows that RBPs Celf1 and Elavl1 are necessary for precise spatiotemporal expression of TFs in the lens. This bridges an important knowledge-gap on the mechanism of how factors such as Pax6, whose dosage in critical to eye development, are regulated on the post-transcriptional level in the lens.

This is a 2020 ARVO Annual Meeting abstract.