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Salma Al Saai, Hayes W McDonald, Kevin Schey, Salil Lachke; Identification of RNA targets and protein binding partners of Tdrd7 in the mouse lens. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1721.
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© ARVO (1962-2015); The Authors (2016-present)
Deficiency of TDRD7 (Tudor domain containing protein 7) causes cataracts in human, mouse and chicken. Tdrd7, a component of RNA-protein complexes, is involved in post-transcriptional control of gene expression in cell differentiation. While the Tudor domains in Tdrd7 are predicted to interact with methylated arginines or lysines in other proteins, its LOTUS domains are predicted to bind RNA. Tdrd7 germline knockout mice exhibit lens fiber cell defects and mis-regulation of several fiber-expressed factors. However, the mechanism of Tdrd7-mediated regulation in the lens remains unclear. For example, the Tdrd7-interacting components in the lens are not defined. To gain insights into Tdrd7 function, this study focused on the identification of its RNA targets and protein binding partners in the mouse lens.
Lenses were isolated from wild-type ICR mice at postnatal day 8 for use in immunoprecipitation (IP) assays with Tdrd7-specific antibody or IgG antibody (control). The IP was subjected to mass spectrometry analysis for identification of proteins that were present in the Tdrd7 pull-down complex. For RNA immunoprecipitation (RIP) assay, RT-qPCR was performed after Tdrd7-IP.
Investigation of Tdrd7-protein partners by mass spectrometry detected 8 proteins that had at least two independent polypeptides recognized at <5% FDR specifically in the Tdrd7-IP, but not in the control. Tdrd7 was detected in the test but not in the control, indicative of the specificity of the pull-down. The other candidate proteins identified in Tdrd7-IP but not in control are: Tubb4b (Tubulin, beta 4B), Tuba1b (Tubulin, alpha 1B), Eef1a1 (Eukaryotic translation elongation factor 1 alpha 1), Utp20 (Small subunit (SSU) processome component 20), Crygc (Gamma crystallin C), Crygs (Gamma crystallin S), Crybb2 (Beta crystallin B2). Interestingly, Utp20 and Eef1a1 are involved in rRNA-processing and translation, respectively. Investigation of Tdrd7-target RNAs by RIP assay identified enrichment of Hsbp1 (Heat shock protein b1) and Actn2 (Actinin alpha 2) mRNAs, both of which are down-regulated in Tdrd7-/- mouse lenses, in turn suggesting the direct control of these factors by Tdrd7.
We have investigated Tdrd7-target transcripts and protein-binding partners in mouse lens. In addition to its potential involvement in protein translation, these data suggest that Tdrd7 functions in the control of lens fiber cytoskeleton.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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