Phenotypic and molecular characterization of cataract in the TDRD7 knockout mouse mutant
Date
2015
Authors
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Publisher
University of Delaware
Abstract
We recently demonstrated that mutations in a conserved RNA granule component Tudor geneTDRD7 cause congenital or juvenile cataracts in humans. Although several distinct classes of cytoplasmic RNA granules that mediate post-transcriptional gene expression control are identified in metazoan cells, their significance in cellular differentiation, especially during mammalian development, is unclear. Moreover, the function of TDRD-family proteins beyond the germline is not characterized. Here, we conducted a comprehensive set of genetic, molecular, cellular and in silico analyses to elucidate the molecular pathogenic mechanism of cataracts in a Tdrd7 targeted germline knockout (KO) mouse model that closely phenocopies the human defect. Bright-field imaging, histology, and scanning electron microscopy-based analyses demonstrate that Tdrd7 KO lenses appear normal at post-natal day (P) 15, before precipitously exhibiting fiber cell defects at P18, and severe cataracts at P22. RNA-sequencing combined with a downstream integrated analysis, and protein 2-D fluorescence difference gel electrophoresis coupled with mass spectrometry, identified two candidates Actn2 and Hspb1 involved in F-actin deposition, which are down-regulated inTdrd7 KO lenses. Indeed, whole mount phalloidin staining demonstrates that F-actin deposition is severely disrupted in mutant lenses. Further, co-immunostaining indicates that TDRD7 granules closely associate with ATXN2 granules in fiber cells and RNA immunoprecipitation demonstrates that TDRD7 directly associates with Actn2 and Hspb1 mRNAs in the lens. Based on this data, we propose a model in which TDRD7 functions with a second RG component protein in post-transcriptional regulation of lens-expressed mRNAs to establish and maintain fiber cell cytoskeleton, disruption of which leads to cataracts.