Abstract
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.
Highlights
Perturbations in lens development results in congenital cataract in humans and animal models (Graw, 2009; Shiels and Hejtmancik, 2019)
While the mRNAs misexpressed in Tdrd7-/- mouse lens have been examined on the genomic level in previous studies (Lachke et al, 2011; Barnum et al, 2020), there is no information on the impact of Tdrd7 deficiency on lens miRNA expression
The stage P4 was selected for this analysis because it preceded cataract formation in Tdrd7-/- lenses (Figure 1D) and was considered to yield information on the early alterations in miRNA expression, prior to the onset of lens defects, in turn reducing the possibility of detecting secondary miRNA changes
Summary
Perturbations in lens development results in congenital cataract in humans and animal models (Graw, 2009; Shiels and Hejtmancik, 2019). Very few RBPs and posttranscriptional regulatory factors, including miRNAs, have been functionally implicated in lens development and cataract (Lorén et al, 2009; Lachke et al, 2011; Choudhuri et al, 2013; Shaham et al, 2013; Wolf et al, 2013; Xie et al, 2014; Dash et al, 2015, 2020; Siddam et al, 2018; Aryal et al, 2020; Barnum et al, 2020; Nakazawa et al, 2020; Shao et al, 2020) This limited information highlights a substantial knowledge-gap in lens and cataract research because post-transcriptional control represents critical mechanisms that allow precise calibration, in terms of dosage and spatiotemporal pattern, of the cellular proteome. These regulatory mechanisms may be significant for controlling mRNA and protein abundance in lens fiber cells – a cell fate that faces added challenges to regulate these basic processes as they undergo nuclear degradation in terminal differentiation (Dash et al, 2016)
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