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Sandeep Aryal, Archana D Siddam, Salil Anil Lachke; Investigation of proteins interacting with the cataract-linked RNA-binding protein Celf1 in mouse lens development. Invest. Ophthalmol. Vis. Sci. 2018;59(9):895. doi: https://doi.org/.
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Deficiency of the conserved RNA-binding protein (RBP) Celf1 causes lens defects and cataract in vertebrates. Celf1 regulates the proteome by distinct post-transcriptional control (PTC) mechanisms such as pre-mRNA splicing, mRNA decay, and translational control. These Celf1-mediated PTC outcomes depend on its interaction with other regulatory proteins. However, the specific protein interactors of Celf1 that determine its function in lens have not been investigated. Here we report novel Celf1-interacting proteins that offer new insights in Celf1-mediated PTC in mouse lens.
Immunoprecipitation (IP) was performed with Celf1-specific antibody (test) or IgG (control) on protein lysates prepared from embryonic day (E) 16.5 and early post-natal day (P) 5 wild-type mouse lens, and the mouse lens epithelium-derived cell line 21EM15. High-throughput tandem mass spectrometry (MS/MS) was then performed using Q Exactive LC-MS/MS system. The acquired spectra were searched against Uniprot mouse proteome database using Sequest and data was analyzed using Proteome Discoverer v1.4.
Using MS/MS and customized prioritization criteria, we identified 79 high-priority candidate proteins in the Celf1-pulldown from 21EM15 lens cell line and E16.5 and P5 lens lysates. These proteins include lens expressed RBPs that are identified in the iSyTE 2.0 database. Functional annotation clustering analysis identified several RBPs (Elavl2, Elavl3, Hnrnpk, Hnrnpd), splicing factors (Prpf4, Sf3b3, Snrnp40), translation factors (Eif3g, Eif4a2, Eife3) and ribonucleases (Pan3, RnaseI). Expression of Elavl2, Hnrnpk, Prpf4 and Sf3b3 in lens tissue was validated by Western blot. Interestingly, Elavl2 and Igf2bp3 are shown to form a ribonucleoprotein complex with Celf1 to mediate RNA localization in vegetal cortex of Xenopus oocyte, suggesting a new direction for exploring Celf1 function in the mouse lens.
Celf1-IP coupled with MS/MS-based proteomic analysis identified potential new Celf1-binding proteins in embryonic, postnatal mouse lens and 21EM15 cell line. They confirm two Celf1-interactors (Elavl2, Igf2bp3) previously identified in non-lens tissues. These data define the Celf1 protein-protein interactome in the lens, in turn providing new directions for investigating RBP-based combinatorial control on the post-transcriptional level in lens development, and their relevance to cataract.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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