The Cataract‐associated RNA‐Binding Protein Celf1 Post‐transcriptionally Regulates the Key Eye Transcription Factor Pax6

Abstract

The lens is a transparent tissue that focuses light on the retina allowing high‐resolution vision. Loss of lens transparency is defined as cataract, the leading cause of blindness worldwide. An estimated 25–50% of congenital cataract cases arise from underlying genetic alterations, making it important to characterize cataract‐linked genes and their pathological mechanisms.Recently, our lab demonstrated that deficiency of the gene Celf1 causes cataract in fish and mouse, indicating its conserved function in vertebrate lens development. Celf1 encodes an RNA‐binding protein (RBP) that directly binds to specific mRNAs and control them on multiple “post‐transcriptional” levels, impacting their alternative splicing, decay or translation into protein. An important unresolved question is whether a post‐transcriptional regulator like Celf1 controls key transcription factors (TFs) in lens development. To address this fundamental question, I generated and characterized lens‐specific Celf1 conditional knockout mice (Celf1cKO/lacZKI). Using immunofluorescence assays, I demonstrate that Celf1cKO/lacZKI lenses exhibit abnormally high protein levels of the homeodomain‐TF Pax6. Further, by performing Celf1‐antibody coupled RNA‐immunoprecipitation (RIP) followed by RT‐qPCR, I demonstrate that Celf1 protein directly associates with Pax6 mRNA.Next, I investigated if Celf1 coordinately functions with other RBPs to exert combinatorial control over gene expression. I performed co‐immunoprecipitation to demonstrate that Celf1 is associated with another RBP, specifically HuR/Elavl1 in lens cells. Finally, by performing HuR/Elval1‐antibody coupled RIP‐RT‐qPCR assay I show that HuR/Elavl1 binds to Pax6 mRNA.These data indicate that the RBPs Celf1 and HuR/Elavl1 directly interact and bind to Pax6 mRNA to mediate combinatorial control of Pax6 protein expression in mouse lens development. Alterations of Pax6 levels are known to cause severe eye defects in human and animal models. Thus, my research uncovers a novel RBP‐based molecular mechanism for regulation of Pax6 expression in the lens, in turn, informing on a new pathway for the pathology of eye defects, including cataract.Support or Funding InformationNEI/NIH RO1 EY021505, Milton H. Stetson Memorial Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CELF1 promotes matrix metalloproteinases gene expression at transcriptional level in lens epithelial cells

“…To achieve lens transparency, Celf1 regulates key factors, such as p27 Kip1 and Dnase2b which are necessary for degradation of nuclear and fiber cell morphology by controlling the expression level of target mRNAs, providing new Celf1-regulated mechanisms involved in lens transparency. Using immunofluorescence assays, abnormally high expression of Pax6 protein was observed in CELF1 knockdown lenses compared to control lenes, suggesting that the eye transcription factor PAX6 is a key target regulated by CELF1 in lens development [19]. In addition, Aryal et al [20] found that Celf1 could post-transcriptionally control the spatiotemporal expression of the key homeodomain transcription factors Pax6 and Prox1 in the lens development in mice.…”