Identification And Characterization Rna Binding Proteins Involved In Post Transcrptional Control Of Caenorhabditis Elegans Germline Development PDF Download

Eye development in vertebrates is initiated in late gastrulation and involves coordinated morphogenesis between the optic vesicle and the non-neural surface ectoderm resulting in the formation of the neural retina and the lens, respectively. While transcription and signaling events required for eye development are well understood, post-transcriptional control of gene expression, especially mediated by RNA-binding proteins (RBPs) is less clear. This represents a significant knowledge-gap as RBPs are important regulatory molecules in the cell that can control the fate of their target mRNAs by interacting with them throughout the mRNA life-cycle and mediating their processing, intra-cellular transport and localization, stability, translation into protein, and ultimately, their degradation. This is also a significant knowledge gap because there are similar number of RBPs encoded by the human genome as there are transcription factors, but the former class of proteins are not as well understood in the context of organogenesis and birth defects as compared to the latter. ☐ While high-throughput sequencing has identified several RBPs to be expressed in the eye, the functional significance in eye development for the vast majority of these factors is yet to be determined. Recently, the Lachke laboratory has identified two conserved RBPs required for eye development, Tdrd7 and Celf1, whose deficiency in the lens results in cataract in vertebrates. To further investigate the importance of RBP-mediated post-transcriptional gene expression control in eye development, I applied a systems-based bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to identify two new RBPs, Rbm24 and Caprin2, which are enriched during early mouse lens development, but whose molecular function in eye development had thus far not been determined. In this research dissertation, I have characterized the function of both Rbm24 and Caprin2 using constitutive and conditional targeted gene deletion mouse models. Further, in collaboration with Dr. Diane Slusarski’s laboratory (University of Iowa), zebrafish rbm24a knockout (by CRISPR/Cas9) and knockdown (by morpholino) mutants were generated and characterized. Together, these findings have led to a comprehensive understanding of the function of these RBPs in vertebrate eye development. ☐ Rbm24-targeted deletion in mouse and rbm24a-CRISPR/Cas9-targeted knockout or morpholino-knockdown in zebrafish causes the developmental defects microphthalmia (small eye) or anophthalmia (no eye). Rbm24 deficiency leads to apoptotic defects in the mouse ocular tissue as well as downregulation of eye development markers such as Sox2, Lhx2, Jag1, E-cadherin and g-Crystallins. Further, similar to the observations in the mouse, sox2 expression is also found to be reduced in rbm24a-morphant zebrafish, indicating the conservation of the Rbm24-Sox2 regulatory module in vertebrate eye development. About 20% of human anophthalmia cases are linked to SOX2 mutations alone. Therefore, I focused on investigating the post-transcriptional molecular mechanism of Rbm24-mediated Sox2 regulation. Sox2 is an intronless gene whose encoded mRNA contains AU-rich regions (ARE) in its 3’UTR. Interestingly, Rbm24 is known to bind to ARE sites in target mRNA. Therefore, to test if Rbm24 directly binds to Sox2 mRNA in vitro and in vivo, I performed RNA-Electrophoretic Mobility Shift assay (EMSA) and RNA-Immunoprecipitation (RIP), respectively. RNA-EMSA showed that Rbm24 protein directly binds to a 20 bp oligomer based on the mouse Sox2 mRNA sequence, and that an intact ARE is necessary for this protein-RNA binding. In turn, RIP assay on E14.5 wildtype mouse ocular tissue suggests that Rbm24 directly binds to Sox2 mRNA in vivo in eye development. To understand the biological significance of this direct Rbm24 protein-Sox2 mRNA molecular interaction, I performed an RNA-decay assay in NIH3T3 cells by co-transfected them with an Rbm24-overexpression vector and a Renilla luciferase reporter vector. In this assay, the Renilla luciferase gene ORF (open reading frame) is fused with the mouse Sox2 mRNA 3’UTR, which contains the three intact ARE sites, and reporter transcripts were quantified after Actinomycin-D treatment to transfected cells. This analysis demonstrates that in conditions of Rbm24 over-expression, the intact Sox2 3’UTR can render increased stability to the reporter transcript. Thus, Rbm24 positively controls Sox2 expression by binding to ARE sites in its 3’UTR and increasing its mRNA stability. Further, mutation analysis in the RNA-decay assay extends the in vitro observation that the binding of Rbm24 to the Sox2 mRNA 3’UTR depends on ARE by providing in vivo evidence that the presence of the ARE sites is necessary for the stability effect rendered by the Sox2 mRNA 3’UTR upon Rbm24 overexpression. Further, because Sox2 is one of the original four Yamanaka pluripotency/cellular reprogramming factor (along with Oct4, Klf4 and c-Myc), I investigated the impact of Rbm24 on the expression of other reprogramming factors such as Oct4, Klf4, c-Myc as well as, Nanog, another established pluripotency factor. I find that over-expression of Rbm24 in several different cell lines such as NIH3T3 (mouse embryo fibroblast cell line), 21EM15 (mouse lens epithelial cell line) and C2C12 (mouse myoblast cell line) results in the up-regulation of Sox2, Oct4 and Klf4. Further, in Rbm24-overexpressed C2C12 cells, Nanog and c-Myc are also upregulated. These data highlight that Rbm24 mediates post-transcriptional control of key transcription and pluripotency factors in vertebrate development. ☐ To gain insight into the function of the other newly identified RBP, Caprin2, in lens biology, I first performed expression analysis of Caprin2 in mouse lens development using in situ hybridization, western blotting and immunostaining. These experiments validate the iSyTE prediction that Caprin2 mRNA and protein are highly expressed and enriched in mouse embryonic and postnatal lens. I generated lens-specific Caprin2 conditional knockout (cKO) mouse mutants using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2cKO/cKO mice, wherein Caprin2 is expected to be deleted in the lens starting from E9.5 due to Cre-mediated re-arrangement of the Caprin2 alleles, revealed two distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2cKO/cKO mutants have an abnormally compact “lens nucleus”, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Further, at a reduced penetrance (8%), I find that Caprin2cKO/cKO mutants exhibit an ocular defect wherein the lens and the cornea remain attached by a persistent stalk, resembling the human developmental defect termed Peters anomaly. These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development. ☐ Together the findings in this dissertation have demonstrated that conserved RBPs such as Rbm24 and Caprin2 have evolved distinct functions in vertebrate eye development and their deficiency leads to microphthalmia and anophthalmia, and lens defects and Peters anomaly, respectively, thus impacting the study of ocular defects in humans.