Browsing by Author "Barnum, Carrie E."
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Item Genome-Wide Analysis of Differentially Expressed miRNAs and Their Associated Regulatory Networks in Lenses Deficient for the Congenital Cataract-Linked Tudor Domain Containing Protein TDRD7(Frontiers in Cell and Developmental Biology, 2021-02-16) Anand, Deepti; Al Saai, Salma; Shrestha, Sanjaya K.; Barnum, Carrie E.; Chuma, Shinichiro; Lachke, Salil A.Mutations/deficiency of TDRD7, encoding a tudor domain protein involved in post-transcriptional gene expression control, causes early onset cataract in humans. While Tdrd7 is implicated in the control of key lens mRNAs, the impact of Tdrd7 deficiency on microRNAs (miRNAs) and how this contributes to transcriptome misexpression and to cataracts, is undefined. We address this critical knowledge-gap by investigating Tdrd7-targeted knockout (Tdrd7-/-) mice that exhibit fully penetrant juvenile cataracts. We performed Affymetrix miRNA 3.0 microarray analysis on Tdrd7-/- mouse lenses at postnatal day (P) 4, a stage preceding cataract formation. This analysis identifies 22 miRNAs [14 over-expressed (miR-15a, miR-19a, miR-138, miR-328, miR-339, miR-345, miR-378b, miR-384, miR-467a, miR-1224, miR-1935, miR-1946a, miR-3102, miR-3107), 8 reduced (let-7b, miR-34c, miR-298, miR-382, miR-409, miR-1198, miR-1947, miR-3092)] to be significantly misexpressed (fold-change ≥ ± 1.2, p-value < 0.05) in Tdrd7-/- lenses. To understand how these misexpressed miRNAs impact Tdrd7-/- cataract, we predicted their mRNA targets and examined their misexpression upon Tdrd7-deficiency by performing comparative transcriptomics analysis on P4 and P30 Tdrd7-/- lens. To prioritize these target mRNAs, we used various stringency filters (e.g., fold-change in Tdrd7-/- lens, iSyTE-based lens-enriched expression) and identified 98 reduced and 89 elevated mRNA targets for overexpressed and reduced miRNAs, respectively, which were classified as “top-priority” “high-priority,” and “promising” candidates. For Tdrd7-/- lens overexpressed miRNAs, this approach identified 18 top-priority reduced target mRNAs: Alad, Ankrd46, Ceacam10, Dgat2, Ednrb, H2-Eb1, Klhl22, Lin7a, Loxl1, Lpin1, Npc1, Olfm1, Ppm1e, Ppp1r1a, Rgs8, Shisa4, Snx22 and Wnk2. Majority of these targets were also altered in other gene-specific perturbation mouse models (e.g., Brg1, E2f1/E2f2/E2f3, Foxe3, Hsf4, Klf4, Mafg/Mafk, Notch) of lens defects/cataract, suggesting their importance to lens biology. Gene ontology (GO) provided further insight into their relevance to lens pathology. For example, the Tdrd7-deficient lens capsule defect may be explained by reduced mRNA targets (e.g., Col4a3, Loxl1, Timp2, Timp3) associated with “basement membrane”. GO analysis also identified new genes (e.g., Casz1, Rasgrp1) recently linked to lens biology/pathology. Together, these analyses define a new Tdrd7-downstream miRNA-mRNA network, in turn, uncovering several new mRNA targets and their associated pathways relevant to lens biology and offering molecular insights into the pathology of congenital cataract.Item Phenotypic and molecular characterization of cataract in the TDRD7 knockout mouse mutant(University of Delaware, 2015) Barnum, Carrie E.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.