Siddam AD , Gautier-Courteille C , Perez-Campos L , Anand D , Kakrana A , Dang CA , Legagneux V , Méreau A , Viet J , Gross JM , Paillard L , Lachke SA .

R01EY021505 NIH HHS , P50-NS40828 NIH HHS , 1U54HD090255 NIH HHS , P20 GM103446 NIH HHS , R01 EY021505 NEI NIH HHS , P20 GM103446 NIGMS NIH HHS , S10 RR027273 NCRR NIH HHS , P50 NS040828 NINDS NIH HHS , U54 HD090255 NICHD NIH HHS

	Fig 1. Celf1 is required for vertebrate lens development.(A) In zebrafish, celf1 transcripts are detected in the lens at 1 day post fertilization (1dpf) by in situ hybridization (ISH). (B) In X. laevis, ISH indicates strong celf1 expression (arrow) in the embryonic St. 30 eye (arrow) and lens (indicated by broken line area). (C) In mouse, ISH shows strong Celf1 transcript expression in the lens at embryonic day 12.5. (D) In mouse lens, Celf1 protein is expressed at (D) E11.5 (E) E14.5 and (F) E16.5 predominantly in fiber cells (f) and to a lower extent in epithelial cells (e). (G, G’) In zebrafish, while control eyes are normal, celf1 knockdown (KD) results in microphthalmia and clouding of lens (asterisk) by 4dpf. (H, H’) In X. laevis, compared to control, celf1 KD results in microphthalmia. (I, I’) In mouse, compared to control, Celf1cKO/cKO lens exhibits severe cataract (asterisk). (J-K’) Compared to control, refraction errors (asterisk) are observed in Celf1cKO/cKO lens under dark-field and light-field microscopy. (L, L’) At E16.5 stage, the mouse Celf1cKO/cKO lens exhibits abnormal spaces (asterisk) in the fiber cell region. Scale bar in F is 75 μm.
	Fig 2. Celf1cKO/lacZKI mouse exhibits mis-expression of key lens genes.(A) Microarray heat-maps representing genes mis-regulated in Celf1cKO/lacZKI lenses compared to control (left column, ±2.5 fold-change, p<0.05, total 34 genes, indicated by heatmap color gradients (left columns: green, down in Celf1cKO/lacZKI; red, up in Celf1cKO/lacZKI) and their respective enrichment in normal lens compared to whole-embryonic tissue as per iSyTE (right columns, lens-enrichment in fold-change indicated by red color intensity). (B) Differentially expressed genes (DEGs) in Celf1cKO/lacZKI lenses are plotted on the X-axis as down-regulated (circles) and up-regulated genes (triangles). On the Y-axis, DEGs are separated based on their lens-enrichment. Red and green color gradients represent high and low lens-enrichment, respectively. Genes down-regulated in Celf1cKO/lacZKI lenses are predominantly highly-lens enriched, while those up-regulated do not exhibit this trend.
	Fig 3. Celf1 deficiency in mouse and fish causes fiber cell nuclear degradation defects.(A, B) Histological analysis of control and Celf1lacZKI/lacZKI mouse lenses at post natal day 4 (P4) stage shows abnormal presence of nuclei in centrally located fiber cells only in Celf1lacZKI/lacZKI mice. (A’, B’) High-magnification of the dotted-line area in A, B. Asterisk denote abnormally retained nuclei. (C, D) In zebrafish, compared to control, celf1KD lens exhibit abnormal presence of nuclei in the central fiber cell region. (C’, D’) High-magnification of the dotted-line area in E, F. Asterisk denote abnormally retained nuclei. (E) RT-qPCR analysis confirms significant Dnase2b down-regulation in Celf1cKO/lacZKI lenses compared to control. (F) RNA immunoprecipitation (RIP) and (G) cross-linked RNA immunoprecipitation (CLIP) shows Dnase2b to be enriched in Celf1-pulldown in wild-type mouse lens. (H) Celf1 over-expression in NIH3T3 cells, which carry Dnase2b 3’UTR downstream of a luciferase reporter, results in significant increase of luciferase mRNA. Abbr.: f.c., fold-change; NS, not significant. Asterisks in E, G, H indicate a p-value < 0.005.
	Fig 4. Celf1-mediated post-transcriptional control of mitotic machinery components facilitates lens fiber cell nuclear degradation.(A, A’) Compared to control lens, phosphorylation of Lamin A/C (pLamin A/C) is reduced in Celf1cKO/lacZKI lens. Dotted-line areas of the fiber cell regions in control (B-D) and Celf1cKO/lacZKI mice (B’-D’) lenses are shown at high-magnification. Arrows in B, C indicate examples of high pLamin A/C expressing nuclei, while no nuclei are observed in D as this area represents a normal nuclear-free zone in the control lens. Asterisks in B’-D’ indicate reduced signals of pLamin A/C in Celf1cKO/lacZKI lens fiber cell nuclei. Note the presence of nuclei in D’ in the centrally located fiber cells due to the nuclear degradation defects in the Celf1cKO/lacZKI lens. (E, E’) Unlike in the control lens where p27Kip1 protein is restricted to cells of the transition zone and cortical fiber cells (arrow), high levels of p27Kip1 protein are detected in the entire fiber cell compartment (asterisks) including the central region in Celf1cKO/lacZKI lens. (F, F’) High-magnification of dotted-line areas in E and F. Asterisks indicate elevated signals of p27Kip1 protein. (G) Quantification of the p27Kip1 immunofluorescence signals from control and Celf1cKO/lacZKI lenses shows significantly increased p27Kip1 protein levels in Celf1cKO/lacZKI lenses. (H) Western blot analysis shows increased levels of p27Kip1 protein in Celf1cKO/lacZKI lens compared to control. (I) Compared to control, p27Kip1 mRNA levels are not significantly altered in Celf1cKO/lacZKI lens. (J, K) RIP and CLIP assays, respectively, identify p27Kip1 mRNA to be highly enriched in Celf1-pulldown in wild-type mouse lens. (L) A potential Celf1 binding GU-rich region in present in the mouse p27Kip1 5’ UTR. (M) Activity of firefly luciferase fused downstream of p27Kip1 5’ UTR is significantly elevated in Celf1-knockdown (Celf1-KD) mouse lens cell line compared to control (firefly luciferase normalized to Renilla luciferase (Rluc)). (N-O’) Compared to control, p21Cip1 mRNA and protein is abnormally elevated in Celf1cKO/lacZKI lens. Asterisk in O’ indicate high expression of p21Cip1 protein in the Celf1cKO/lacZKI lens. Abbr.: e, epithelial cells; f, fiber cells; tz, transition zone; NS, not significant. Scale bar in A’, E’, O’ is 75 μm and in D’ and F’ is 12 μm. Asterisks in G, K, M and N indicate a p-value < 0.0005, 0.005, 0.05 and 0.005, respectively.
	Fig 5. Celf1 deficiency in mouse and fish causes defects in fiber cell morphology.(A) RT-qPCR analysis confirms significant Actn2 down-regulation in Celf1cKO/lacZKI lenses compared to control. (B) RNA immunoprecipitation (RIP) identifies Actn2 as an enriched transcript in Celf1-pulldown in P15 wild-type mouse lens. (C) Cross-linked RNA immunoprecipitation (CLIP) shows Sptb transcripts to be enriched in Celf1-pulldown in wild-type mouse lens. (D) RT-qPCR analysis shows that the high-abundant Sptb isoform (isoform 1 (ENSMUST00000021458)) is reduced, while the low-abundant Sptb isoform (isoform 2 (ENSMUST00000166101)) is abnormally elevated in Celf1cKO/lacZKI lenses. (E, E’) In mouse, phalloidin staining of lens tissue sections (stage P0) shows uniform F-actin deposition along the fiber cell margins in control, while Celf1cKO/lacZKI lenses exhibit abnormal pattern of F-actin (asterisk). (F, F’) In zebrafish, while control lens exhibits normal F-actin deposition, celf1KD lens (stage 4dpf) exhibits abnormal F-actin pattern (asterisk) in fiber cells. (G-H’) In mouse, scanning electron microscopy analysis of cortical and nuclear fiber cells shows disrupted cell organization (asterisk) in Celf1cKO/lacZKI lenses (stage P15). Scale bar in D’ is 75 μm and G’ is 2.5μM.
	Fig 6. Model for Celf1-mediated post-transcriptional gene expression control in the lens.In normal lens development, Celf1 is required for nuclear degradation and proper cell morphology in fiber cell differentiation. Celf1 positively regulates the nuclease Dnase2b (being necessary for its high mRNA levels) and negatively regulates the cyclin-dependent kinase inhibitors p21Cip1 (being necessary for its low mRNA levels) and p27Kip1 (by inhibiting its translation into protein). Inhibition of p21Cip1 and p27Kip1 allows the activation of Cdk1, which phosphorylates Lamin A/C to initiate nuclear envelope breakdown in fiber cells. Thus, Celf1 controls the nuclease (Dnase2b) as well as its access to nuclear DNA, to regulate nuclear degradation in lens fiber cells. These findings show how mitotic machinery components–normally involved in nuclear envelope disassembly during cell division–are post-transcriptionally rewired by RNA-binding proteins to regulate cell differentiation in lens development. Additionally, Celf1 controls the splice isoform abundance of the membrane-organization protein Sptb (β-spectrin) and high transcript levels of the F-actin-binding protein Actn2 (α-actinin 2), to regulate fiber cell morphology. Abbr.: Epi, epithelium; TZ, transition zone; FC, fiber cells.

Agrawal, Compound mouse mutants of bZIP transcription factors Mafg and Mafk reveal a regulatory network of non-crystallin genes associated with cataract. 2015, Pubmed

Agrawal, Compound mouse mutants of bZIP transcription factors Mafg and Mafk reveal a regulatory network of non-crystallin genes associated with cataract. 2015, Pubmed
Anand, An integrative approach to analyze microarray datasets for prioritization of genes relevant to lens biology and disease. 2015, Pubmed
Audette, Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression. 2016, Pubmed
Baker, p21 both attenuates and drives senescence and aging in BubR1 progeroid mice. 2013, Pubmed
Barreau, Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions. 2006, Pubmed
Beaumont, Mutation in the iron responsive element of the L ferritin mRNA in a family with dominant hyperferritinaemia and cataract. 1995, Pubmed
Bennett, Spectrin- and ankyrin-based membrane domains and the evolution of vertebrates. 2013, Pubmed
Brinegar, Roles for RNA-binding proteins in development and disease. 2016, Pubmed
Cavalheiro, N-myc regulates growth and fiber cell differentiation in lens development. 2017, Pubmed
Chaffee, Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly. 2014, Pubmed
Chaudhury, CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT. 2016, Pubmed
Cheng, The lens actin filament cytoskeleton: Diverse structures for complex functions. 2017, Pubmed
Cvekl, Signaling and Gene Regulatory Networks in Mammalian Lens Development. 2017, Pubmed
Cvekl, The cellular and molecular mechanisms of vertebrate lens development. 2014, Pubmed
Dasgupta, The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins. 2012, Pubmed
Dash, RNA-binding proteins in eye development and disease: implication of conserved RNA granule components. 2016, Pubmed
Dash, Deficiency of the RNA binding protein caprin2 causes lens defects and features of Peters anomaly. 2015, Pubmed
De Maria, DNase IIbeta distribution and activity in the mouse lens. 2007, Pubmed
Edwards, Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1. 2013, Pubmed , Xenbase
Gareau, p21(WAF1/CIP1) upregulation through the stress granule-associated protein CUGBP1 confers resistance to bortezomib-mediated apoptosis. 2011, Pubmed
Gautier-Courteille, EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation. 2004, Pubmed , Xenbase
Gerstberger, A census of human RNA-binding proteins. 2014, Pubmed
Giudice, Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development. 2014, Pubmed
Gupta, α-Actinin-2 deficiency results in sarcomeric defects in zebrafish that cannot be rescued by α-actinin-3 revealing functional differences between sarcomeric isoforms. 2012, Pubmed
Göpfert, Cell cycle-dependent translation of p27 involves a responsive element in its 5'-UTR that overlaps with a uORF. 2003, Pubmed
Iakova, Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate. 2004, Pubmed
Jiang, Cap-independent translation through the p27 5'-UTR. 2007, Pubmed
Kakrana, iSyTE 2.0: a database for expression-based gene discovery in the eye. 2018, Pubmed
Kress, Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermatogenesis defects in mice. 2007, Pubmed
Kullmann, ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR. 2002, Pubmed
Kwan, The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. 2007, Pubmed
Lachke, Mutations in the RNA granule component TDRD7 cause cataract and glaucoma. 2011, Pubmed
Lachke, iSyTE: integrated Systems Tool for Eye gene discovery. 2012, Pubmed
Lachke, RNA Granules and Cataract. 2011, Pubmed
Le Tonquèze, Identification of CELF1 RNA targets by CLIP-seq in human HeLa cells. 2016, Pubmed
Le Tonquèze, Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down-regulation. 2010, Pubmed
Lee, Caspase remodeling of the spectrin membrane skeleton during lens development and aging. 2001, Pubmed
Lyu, Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract. 2016, Pubmed
Manning, The roles of RNA processing in translating genotype to phenotype. 2017, Pubmed
Manthey, Development of novel filtering criteria to analyze RNA-sequencing data obtained from the murine ocular lens during embryogenesis. 2014, Pubmed
Marquis, CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding. 2006, Pubmed , Xenbase
Millard, A U-rich element in the 5' untranslated region is necessary for the translation of p27 mRNA. 2000, Pubmed
Nakahara, Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens. 2007, Pubmed
Nishimoto, Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens. 2003, Pubmed
Parker, p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. 1995, Pubmed
Philips, Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. 1998, Pubmed
Rattenbacher, Analysis of CUGBP1 targets identifies GU-repeat sequences that mediate rapid mRNA decay. 2010, Pubmed
Reed, An immunohistochemical method for the detection of proteins in the vertebrate lens. 2001, Pubmed
Rowan, Disassembly of the lens fiber cell nucleus to create a clear lens: The p27 descent. 2017, Pubmed
Rowan, Precise temporal control of the eye regulatory gene Pax6 via enhancer-binding site affinity. 2010, Pubmed
Schleicher, Actin-binding proteins are conserved from slime molds to man. 1988, Pubmed
Seritrakul, Expression of the de novo DNA methyltransferases (dnmt3 - dnmt8) during zebrafish lens development. 2014, Pubmed
Singh, The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion. 2015, Pubmed
Terrell, Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes. 2015, Pubmed
Uribe, Immunohistochemistry on cryosections from embryonic and adult zebrafish eyes. 2007, Pubmed
Vlasova, Conserved GU-rich elements mediate mRNA decay by binding to CUG-binding protein 1. 2008, Pubmed
Vlasova-St Louis, CELFish ways to modulate mRNA decay. 2013, Pubmed
Wolf, Identification and characterization of FGF2-dependent mRNA: microRNA networks during lens fiber cell differentiation. 2013, Pubmed
Zhang, Cooperation between the Cdk inhibitors p27(KIP1) and p57(KIP2) in the control of tissue growth and development. 1998, Pubmed
Zheng, CUG-binding protein represses translation of p27Kip1 mRNA through its internal ribosomal entry site. 2011, Pubmed