THE CATARACT-ASSOCIATED RNA-BINDING PROTEIN CELF1 POST-TRANSCRIPTIONALLY REGULATES THE KEY EYE TRANSCRIPTION FACTOR PAX6 IN LENS DEVELOPMENT
Date
2019-05
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Publisher
University of Delaware
Abstract
The lens is a transparent tissue that, together with the cornea, focuses light on the retina, allowing for high-resolution vision. Lens opacification, termed cataract, is the leading cause of blindness worldwide. Cataract can present commonly as age-dependent or less frequently as congenital. An estimated 25-50% of congenital cataract cases arise from underlying genetic or developmental defects. While several cataract-linked genes are known, it is estimated that many more have yet to be been identified. Characterization of these novel cataract-linked genes and their function in the lens will lead to a better understanding of the cataract pathology and can potentially identify novel therapeutic targets for non-surgical intervention. Therefore, our lab has developed and applied a systems-based tool, iSyTE (integrated Systems Tool for Eye gene discovery), to identify new candidate genes associated with lens development and cataract. Recently, iSyTE was used to identify a new cataract-associated gene called Celf1.
Celf1 encodes an RNA-binding protein (RBP) that functions in distinct regulatory mechanisms in the mRNA life cycle, controlling diverse post-transcriptional processes such as mRNA decay or stability, alternative splicing, and translation into protein. Our lab recently demonstrated that deficiency of Celf1 causes lens defects or cataract in fish, frog, and mouse – indicating that it has a conserved function in vertebrate lens development. This report demonstrated that in normal lens development, Celf1 negatively regulates the translation of the mRNA encoding a key cell cycle regulator, namely the cyclin-dependent kinase inhibitor p27Kip1, in maturing fiber cells. This function of Celf1 in reducing p27Kip1 in advanced stage maturing fiber cells facilitates the activation of CDK1, which mediates phosphorylation of nuclear laminA/C proteins, in turn enabling nuclear envelope breakdown. Furthermore, this initial work also showed that Celf1 is required for the increased stability of the mRNA that encodes the nuclease, Dnase2b. Thus, Celf1 controls both, the enzyme involved in DNA degradation, as well as its access to fiber cell nuclear DNA, which ensures removal of fiber cell nuclei, a requirement for lens transparency. Removal of Celf1 perturbs this regulatory module, causing abnormal retention of nuclei in lens fiber cells contributing to lens opacity. However, this elegant model explains only a subset of the molecular changes that contribute to cataract formation in Celf1 deficient animals. There are many other remaining questions, which may have a broader conceptual impact on lens biology. For example, do RBPs such as Celf1, involved in post-transcriptional control, regulate the expression of key DNA-binding transcription factors (TFs) in the lens? Does Celf1 function in coordination with other RBP binding-partners mediate combinatorial control on the expression of such TFs?
My research addresses these fundamental questions by identifying a new regulatory relationship between Celf1 protein and the key lens TF Pax6. I find that Celf1 deficient mouse lenses exhibit abnormal over-expression of Pax6 protein in lens epithelium and fiber cells. Furthermore, compared to normal lenses, wherein Pax6 protein is restricted to early differentiating fiber cells, the area of Pax6 protein expression in Celf1 deficient lenses is abnormally expanded to include advanced stage differentiating fiber cells. This is significant as Pax6 is a key regulator of eye and lens development and precise control over its protein levels are critical to lens and eye biology. For example, aberrant Pax6 dosage causes a range of ocular defects such as a complete absence of eye tissue (anophthalmia), small eye (microphthalmia), aniridia (absence of the iris), and cataract. Ectopic over-expression of Pax6 also causes lens defects. To investigate the effect of Celf1 protein in controlling Pax6 protein dosage, I applied a Cre-loxp-based mouse genetics approach (mouse lines carrying Celf1 floxed allele and the lens-specific Cre driver allele, Pax6GFPCre) to generate Celf1 lens-specific conditional knockout mice (hereafter referred to as Celf1cKO/lacZKI). Using immunofluorescence assays, I demonstrated that Pax6 protein is abnormally high in Celf1cKO/lacZKI lenses compared to control. Interestingly, real-time quantitative polymerase chain reaction (RT-qPCR) demonstrates that Pax6 mRNA levels are not significantly altered in Celf1cKO/lacZKI lenses. Thus, the elevation of Pax6 protein levels without a comparable elevation of Pax6 mRNA suggests a regulatory mechanism functioning on the post-transcriptional level, specifically at the level of translational control. Similar to the Celf1cKO/lacZKI lens, stable Celf1-knockdown (KD) mouse lens-derived epithelial cell lines also show an up-regulation of Pax6 protein compared to control. To gain insight into the molecular mechanism of how Celf1 protein controls Pax6 mRNA translation, I performed Celf1-antibody coupled RNA-immunoprecipitation (RIP) followed by RT-PCR. Celf1 RIP assay demonstrates that Celf1 protein interacts with Pax6 mRNA. Further, cross-linking immunoprecipitation (CLIP) followed by RNA-sequencing (RNA-seq), performed by our collaborator Dr. Luc Paillard, offers independent support that Celf1 protein directly binds to Pax6 mRNA and identifies a potential Celf1 protein binding-site within the Pax6 mRNA 3’ UTR. Further, I show that a partial sequence of the Pax6 3’ mRNA UTR is sufficient in a luciferase assay for Celf1-mediated repression of Pax6 protein translation.
To gain further insight into the molecular mechanism, I next investigated if Celf1 coordinately functions with other RBPs to exert combinatorial control over gene expression of shared mRNA targets. Co-immunoprecipitation performed in cultured
mouse lens epithelial cells demonstrates that Celf1 associates with another RBP, Elavl1. Celf1 and Elavl1 proteins have been shown to coordinately control protein expression of target mRNAs in other systems. Bioinformatics-based analysis identified multiple potential Elavl1-binding sites, including AU-rich elements (AREs), within the Pax6 transcript sequence. By performing Elavl1-antibody RIP coupled with RT-PCR, I show that Elavl1 protein, similar to Celf1 protein, interacts with Pax6 mRNA in the lens.
Together, these data indicate that the RBPs Celf1 and Elavl1 bind Pax6 mRNA and likely mediate combinatorial control over Pax6 mRNA to control the translation of Pax6 protein in mouse lens development. This study uncovers a novel RBP-mediated post-transcriptional control mechanism for spatio-temporal regulation of the protein levels of a key TF Pax6 in the lens, in turn, providing a new molecular pathway underlying the cataract pathology in Celf1-deficient lenses.
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Keywords
Biological sciences, Lens development, CELF1,PAX6