Although majority of the genes linked to early-onset cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. We report on small Maf transcription factors Mafg and Mafk as regulators of several non-crystallin human cataract-associated genes in fiber cells and establish their significance to this disease. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify Mafg and its co-regulators in the lens, and generated various null-allelic combinations of Mafg:Mafk mouse mutants for phenotypic and molecular analysis. By age 4-months, Mafg−/−:Mafk+/− mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of Mafg−/−:Mafk+/− mouse lens reveals severely disorganized fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of Mafg−/−:Mafk+/− lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to cataract. This approach identifies 36 high-priority candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes oxidative stress and mis-regulation of sterol synthesis. These data identify Mafg and Mafk as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to human cataract.

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Background It was recently demonstrated that deficiency of a conserved RNA binding protein (RBP) and RNA granule (RG) component Tdrd7 causes ocular defects including cataracts in human, mouse and chicken, indicating the importance of post-transcriptional regulation in eye development. Here we investigated the function of a second conserved RBP/RG component Caprin2 that is identified by the eye gene discovery tool iSyTE. Results In situ hybridization, western blotting and immunostaining confirmed highly enriched expression of Caprin2 mRNA and protein in mouse embryonic and postnatal lens. To gain insight into its function, lens-specific Caprin2 conditional knockout (cKO) mouse mutants were generated using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2cKO/cKO mutants revealed 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. Additionally, Caprin2cKO/cKO mutants also exhibited at 8% penetrance a developmental defect that resembles a human condition called Peters anomaly, wherein the lens and the cornea remain attached by a persistent stalk. Conclusions These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development.

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a , Polr1c , and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis–Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.

The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1 and Bmp4 are commonly required for their development. In contrast, our understanding of post-transcriptional regulation in eye development and disease, particularly regarding the function of RNA binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila, as well as several vertebrate models such as fish, frog, chicken and mouse. Interestingly, of the 42 RBPs that have been investigated with for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as Processing bodies (P-bodies), Stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate post-transcriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2 and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving post-transcriptional regulatory networks in eye development and disease.

Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans—yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3’UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3’UTR. Further, we demonstrate that Sox2 3’UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24–Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

Lens development involves a complex and highly orchestrated regulatory program. Here, we investigate the transcriptomic alterations and splicing events during mouse lens formation using RNA-seq data from multiple developmental stages, and construct a molecular portrait of known and novel transcripts. We show that the extent of novelty of expressed transcripts decreases significantly in post-natal lens compared to embryonic stages. Characterization of novel transcripts into partially novel transcripts (PNTs) and completely novel transcripts (CNTs) (novelty score ≥ 70%) revealed that the PNTs are both highly conserved across vertebrates and highly expressed across multiple stages. Functional analysis of PNTs revealed their widespread role in lens developmental processes while hundreds of CNTs were found to be widely expressed and predicted to encode for proteins. We verified the expression of four CNTs across stages. Examination of splice isoforms revealed skipped exon and retained intron to be the most abundant alternative splicing events during lens development. We validated by RT-PCR and Sanger sequencing, the predicted splice isoforms of several genes Banf1, Cdk4, Cryaa, Eif4g2, Pax6, and Rbm5. Finally, we present a splicing browser Eye Splicer (http://www.iupui.edu/~sysbio/eye-splicer/), to facilitate exploration of developmentally altered splicing events and to improve understanding of post-transcriptional regulatory networks during mouse lens development.

Abstract Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7−/− lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7–ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.