The RNA-binding protein Celf1 post-transcriptionally controls Pax6 and Prox1 in lens development

Abstract

Purpose : While many cataract-linked genes have been identified thus far, knowledge on the mechanistic basis of their regulation is restricted to the function of a few key transcription factors (TFs) in the lens. Our recent findings have identified several RNA-binding proteins (RBPs), involved in the post-transcriptional control of gene expression, to be important for fiber cell differentiation and development/maintenance of lens transparency. These data pose a fundamental question: Do RBPs control the expression of TFs in the lens? We addressed this critical knowledge-gap by investigating the role of the cataract-linked RBP, Celf1, in post-transcriptional control of the key TFs Pax6 and Prox1 in lens development.

Methods : Lens-specific Celf1 compound conditional deletion mice were generated crossing Pax6GFPCre and Celf1 flox and germline knock-in alleles (termed Celf1^cKO/lacZKI). Immunofluorescence (IF) was performed on lens sections using antibodies against Pax6 (Developmental Studies Hybridoma Bank) and Prox1 (Covance). Immunoprecipitation (IP) was performed with Celf1 antibody followed by high-throughput tandem mass spectrometry (MS/MS) using Q Exactive LC-MS/MS system. Co-IP was performed using Elavl1 antibody on Celf1 pulldown. Celf1 RNA-IP (RIP) and Elavl1-RIP were performed using RIP-kit (Millipore) on post-natal day (P) 15 mouse lens lysates. For cross-linked IP (CLIP), P13 mouse lenses were UV cross-linked and subjected to Celf1-IP. Isolated RNA was subjected to RT-qPCR using gene-specific primers.

Results : IF assays show that Pax6 and Prox1 proteins are abnormally elevated in Cefl1^cKO/lacZKI lens epithelium and fiber cells. However, their mRNA levels are not altered in Cefl1^cKO/lacZKI whole lenses. To gain insight into the mechanism of Celf1-based control over these TFs, we performed Celf1-IP-MS and co-IP to identify and validate the RBP, Elavl1, as its binding partner in lens cells. Further, RIP assays show that Celf1 and Elavl1 bind to Pax6 and Prox1 mRNAs, which is supported by bioinformatics-analysis that predict their binding sites in Pax6 and Prox1 3’ UTRs. Finally, CLIP assays confirmed that Celf1 binds to Pax6 and Prox1 3’ UTRs.

Conclusions : These data uncover a combinatorial mechanism of RBP-based post-transcriptional control of key transcription factors, Pax6 and Prox1, in lens development and provide new insights into the molecular pathology of cataract in Celf1-deficient animals.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.