Rbm24 post-transcriptionally controls key transcription factors in mouse eye development

Abstract

Purpose : Early lens development involves a complex tissue interplay as the optic vesicle interacts with the presumptive lens ectoderm to induce formation of the lens placode. While signaling and transcription-based control of gene expression in early eye development is well understood, the importance of RNA-binding proteins (RBPs; involved in post-transcriptional control) in optic vesicle and lens placode development, and the associated ocular birth defects, anophthalmia, microphthalmia or cataract, is presently unknown – reflecting a significant knowledge-gap. Using iSyTE (integrated Systems Tool for Eye gene discovery), we identified Rbm24 as a new RBP with enriched expression in lens development. We examined iSyTE’s prediction that Rbm24 may have a key role in eye development.

Methods : Rbm24 germline (Rbm24^-/-) and conditional knockout mice (Rax-CreER^T2:Rbm24^flox:flox; termed Rbm24^cKO) were analyzed. RNA-immunoprecipitation (RIP) was performed using Rbm24-specific antibody. EMSA (electrophoretic mobility shift assay) was performed using Rbm24 protein and biotinylated Sox2 3’ UTR probes. Immunofluorescence (IF) was performed for specific proteins.

Results : Rbm24^-/- and Rbm24^cKO mice exhibit fully penetrant early eye defects microphthalmia and anophthalmia, similar to human SOX2-deficiency defects. Rbm24 deficiency leads to reduced expression of Sox2, Lhx2, Pax6, Jag1, E-cadherin and gamma-Crystallins in developing eye tissue. RIP assays show enriched Sox2 and Lhx2 mRNA in the Rbm24 pulldown, suggesting direct interaction. EMSA shows that Rbm24 directly binds to Sox2 mRNA 3'UTR, which is dependent on AU-rich elements (AREs). Further, mRNA decay assays indicate that intact Sox2 3'UTR AREs are necessary for Rbm24-based elevation of Sox2 mRNA half-life. Lhx2 mRNA is significantly downregulated in Rbm24-knockdown cells and upregulated in Rbm24-overexpressed cells. Thus, Rbm24 has a key role in early eye development.

Conclusions : These data demonstrate that a new RBP, Rbm24, is expressed earlier in eye development compared to other previously characterized RBPs. Rbm24 functions in the optic vesicle and lens placode and its deficiency causes anophthalmia and/or microphthalmia. These findings advance our understanding of a new layer of regulatory mechanism controlled by Rbm24 that regulates key eye transcription factors such as Sox2 and Lhx2, which are linked to anophthalmia/microphthalmia in animal models or humans.

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