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

Abstract

Mutations/deficiency of <i>TDRD7</i>, 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 <i>Tdrd7</i> deficiency on microRNAs (miRNAs) and how this contributes to transcriptome misexpression and to cataracts, is undefined. We address this critical knowledge-gap by investigating <i>Tdrd7</i>-targeted knockout (<i>Tdrd7-/-</i>) mice that exhibit fully penetrant juvenile cataracts. We performed Affymetrix miRNA 3.0 microarray analysis on <i>Tdrd7-/-</i> 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, <i>p</i>-value < 0.05) in <i>Tdrd7-/-</i> lenses. To understand how these misexpressed miRNAs impact <i>Tdrd7-/-</i> cataract, we predicted their mRNA targets and examined their misexpression upon <i>Tdrd7</i>-deficiency by performing comparative transcriptomics analysis on P4 and P30 <i>Tdrd7-/-</i> lens. To prioritize these target mRNAs, we used various stringency filters (<i>e.g.</i>, fold-change in <i>Tdrd7-/-</i> 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 <i>Tdrd7-/-</i> lens overexpressed miRNAs, this approach identified 18 top-priority reduced target mRNAs: <i>Alad</i>, <i>Ankrd46</i>, <i>Ceacam10</i>, <i>Dgat2</i>, <i>Ednrb</i>, <i>H2-Eb1</i>, <i>Klhl22</i>, <i>Lin7a</i>, <i>Loxl1</i>, <i>Lpin1</i>, <i>Npc1</i>, <i>Olfm1</i>, <i>Ppm1e</i>, <i>Ppp1r1a</i>, <i>Rgs8</i>, <i>Shisa4</i>, <i>Snx22</i> and <i>Wnk2</i>. Majority of these targets were also altered in other gene-specific perturbation mouse models (<i>e.g., Brg1</i>, <i>E2f1/E2f2/E2f3</i>, <i>Foxe3</i>, <i>Hsf4</i>, <i>Klf4</i>, <i>Mafg</i>/<i>Mafk</i>, <i>Notch</i>) 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 <i>Tdrd7</i>-deficient lens capsule defect may be explained by reduced mRNA targets (<i>e.g., Col4a3</i>, <i>Loxl1</i>, <i>Timp2</i>, <i>Timp3</i>) associated with "basement membrane". GO analysis also identified new genes (<i>e.g., Casz1</i>, <i>Rasgrp1</i>) 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.