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Carrie Ellen Barnum, Shaili Patel, David A Scheiblin, Shawn Polson, Shinichiro Chuma, David C Beebe, Salil Anil Lachke; RNA-sequencing analysis of Tdrd7 mutant lens identifies new differentially regulated targets. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1681.
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© ARVO (1962-2015); The Authors (2016-present)
We recently demonstrated that deficiency of the RNA granule component gene TDRD7 causes cataracts in human, mouse and chicken. Tdrd-family proteins are known to function in post-transcriptional control of gene expression in spermatogenesis. Two different Tdrd7 null mouse mutants have been generated (one was identified in an ENU-screen and the other is a targeted germline knockout (KO) mutant), and both exhibit severe cataracts. To investigate the mechanism of Tdrd7 function in the lens, we performed detailed phenotypic and molecular characterization of Tdrd7 KO mouse mutants.
Phenotypic analysis was performed by light microscopy, scanning electron microscopy (SEM), confocal microscopy and laser capture microdissection. Molecular analysis was performed by running separate RNA-sequencing (RNA-seq) experiments for isolated “large” (mRNA) and “small” (miRNA, piRNA, snoRNA, snRNA) RNA fractions to identify differentially regulated transcripts.
In the first three weeks of life, Tdrd7 KO lenses appear normal in light microscopy until abruptly exhibiting lens defects by postnatal day (P) 22. However, SEM analysis demonstrates that fiber cell defects are discernable in Tdrd7 KO lens at P18, indicating an earlier onset of the phenotype. Large RNA-seq of Tdrd7 KO P4 lens confirmed Hsbp1 (Hsp27) downregulation and identified several new differentially regulated mRNA targets, including other Hsp family members. Small RNA-seq identified piwi-interacting RNAs (piRNAs) to be expressed in the lens, some of which exhibited differential regulation in Tdrd7 KO lens. This is intriguing because piRNAs were previously considered to be restricted to germ cells, where they are regulated by Tdrd-proteins. Additionally, several snoRNAs, snRNAs and miRNAs were found to be differentially regulated in Tdrd7 KO lenses, suggesting a critical function of Tdrd7 in regulating non-protein coding RNAs in the lens.
We used RNA-seq to investigate Tdrd7 KO lens and have identified many new differentially regulated transcripts. Significantly, we have identified piRNAs in the lens and demonstrate that in addition to affecting mRNA profiles, Tdrd7 nullizygosity affects expression of select snoRNA, snRNA, miRNA and piRNA. These data suggest that Tdrd7 plays distinct functions, regulating both mRNAs and non-protein-coding RNAs, to mediate post-transcriptional control of gene expression in lens fiber cells.
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