Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5’ UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.
RT-qPCR of Dnase2b in mouse lens (Fig 3E)
RT-qPCR assay data for Dnase2b mRNA in WT and Celf1 cKO mouse lens (Fig 3E in Siddam et al. 2018 PLOS Genetics)
Fig 3E .xlsx
Celf1 CLIP assay on mouse lens (Fig 3G Fig 4K Fig 5C)
Data for cross-linked immunoprecipitation (CLIP) using Celf1 antibody on wild-type mouse lens followed by RT-qPCR for specific mRNAs: Dnase2b, p27Kip1, Sptb (Fig 3G, Fig 4K, Fig 5C in Siddam et al. 2018 PLOS Genetics)
Fig 3G Fig 4K Fig 5C .xls
Dnase2b 3' UTR-luciferase mRNA assay (Fig 3H)
RT-qPCR data for quantification of luciferase mRNA fused to Dnase2b 3' UTR in cells with Celf1 over-expression (Fig 3H in Siddam et al. 2018 PLOS Genetics)
Fig 3H.xlsx
Quantification of p27Kip1 immunofluorescence in mouse lens (Fig 4G)
Immunofluorescence signal was quantified for p27Kip1 staining in the whole lens area (left) and the central fiber cell area (right) of the lens in control and Celf1 cKO lenses. (Fig. 4G in Siddam et al. 2018 PLOS Genetics)
Fig 4G.xlsx
p27Kip1 mRNA in Celf1 cKO and control mouse lens (Fig 4I)
RT-qPCR data for quantification of p27Kip1 mRNA in control and Celf1 cKO mouse lens (Fig. 4I in Siddam et al. 2018 PLOS Genetics)
Fig 4I.xlsx
Luciferase reporter for p27Kip1 5'UTR in Celf1 knockdown (Fig 4M)
Data for luciferase reporter assay investigating the up-regulation of 5'UTR fused reporter in Celf1 knockdown lens cell line (Fig. 4M in Siddam et al. 2018 PLOS Genetics)
Fig 4M .xlsx
Assay for p21Cip1 mRNA in Celf1 cKO mouse lens (Fig 4N)
RT-qPCR data for p21Cip1 mRNA quantification in control and Celf1 cKO mouse lens (Fig. 4N in Siddam et al. 2018 PLOS Genetics)
Fig 4N.xlsx
Quantification of Actn2 mRNA in Celf1 cKO mouse lens (Fig 5A)
RT-qPCR data for quantification of Actn2 mRNA in control and Celf1 cKO mouse lens (Fig. 5A in Siddam et al. 2018 PLOS Genetics)
Fig 5A.xlsx
Quantification of Sptb isoforms in Celf1 cKO mouse lens (Fig 5D)
RT-qPCR data for quantification of specific Sptb isoforms in control and Celf1 cKO mouse lens (Fig. 5D in Siddam et al. 2018 PLOS Genetics)
Fig 5D.xlsx
iSyTE analysis of Celf1 expression in mouse lens (S1 Fig)
Microarray data values for expression of Celf1 in wild-type mouse lens in three embryonic stages compared to whole embryonic body tissue (Supplementary Fig. S1 in Siddam et al. 2018 PLOS Genetics)
S1 Fig.xlsx
Quantification of Celf1 mRNA in Celf1 cKO mouse lens (S3 Fig)
Data for RT-qPCR quantification of Celf1 mRNA in Celf1 cKO mouse lens compared to control lens (Supplementary Fig. S3 in Siddam et al. 2018 PLOS Genetics)
S3 Fig.xlsx
Quantification of ocular defects in Xenopus celf1-knockdown embryos (S5 Fig)
Data for quantification of ocular phenotypes in celf1-knockdown Xenopus laevis embryos subjected to morpholino-injection on one side (Supplementary Fig. S5 in Siddam et al. 2018 PLOS Genetics)
S5 Fig.xlsx
Quantification of lamin A/C immunofluorescence in Celf1 cKO mouse lens (S11 Fig)
Data for quantification of immunofluorescence signal of lamin A/C in control and Celf1 cKO mouse lens (Supplementary Fig. S11 in Siddam et al. 2018 PLOS Genetics)
S11 Fig.xlsx
