Archana D. Siddam

RNA-binding proteins in eye development and disease: implication of conserved RNA granule components

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 posttranscriptional 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 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, 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 posttranscriptional 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 posttranscriptional regulatory networks in eye development and disease. WIREs RNA 2016, 7:527–557. doi: 10.1002/wrna.1355

An integrative approach to analyze microarray datasets for prioritization of genes relevant to lens biology and disease

Microarray-based profiling represents an effective method to analyze cellular or tissue-specific gene expression on the genome-level. However, in comparative analyses between control and mutant samples, microarrays often identify a large number of differentially expressed genes, in turn making it challenging to isolate the select “high-priority candidates” that are most relevant to an observed mutant phenotype. Here, we describe an integrative approach for mouse mutant lens microarray gene expression analysis using publically accessible systems-level information such as wild-type mouse lens expression data in iSyTE (integrated Systems Tool for Eye gene discovery), protein–protein interaction data in public databases, gene ontology enrichment data, and transcription factor binding profile data. This strategy, when applied to small Maf Mafg −/−:Mafk +/− mouse lens microarray datasets (deposited in NCBI Gene Expression Omnibus database with accession number GSE65500) in Agrawal et al. 2015 [1], led to the effective prioritization of candidate genes linked to lens defects in these mutants. Indeed, from the original list of genes that are differentially expressed at ± 1.5-fold and p < 0.05 in Mafg −/−:Mafk +/− mutant lenses, this analysis led to the identification of thirty-six high-priority candidates, in turn reducing the number of genes for further study by approximately 1/3 of the total. Moreover, eight of these genes are linked to mammalian cataract in the published literature, validating the efficacy of this approach. Additionally, these high-priority candidates contribute valuable information for the assembly of a gene regulatory network in the lens. In sum, the pipeline outlined in this report represents an effective approach for initial as well as downstream microarray expression data analysis to identify genes important for lens biology and cataracts. We anticipate that this integrative strategy can be extended to prioritize phenotypically relevant candidate genes from microarray data in other cells and tissues.

The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development

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.

90 Focus on a clear message: conserved RNA binding proteins function in mRNA control in eye lens development and their deficiency causes cataract

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