This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023. A new in silico approach based on the folding prediction tool AlphaFold2 is now being pursued to identify proteins in the lens that present altered protein-protein interactions between the alternatively spliced proteins isoforms of the cytoskeleton-linked candidates regulated by CELF1. This study reports the application of two omics approaches to identify new AS targets of Celf1 in the lens. Thus, these data lead to the hypothesis that the abnormal splicing of the cytoskeletal protein-encoding RNAs contributes to cataract in Celf1 cKO, by producing protein isoform with an altered interactome. Interestingly, the majority of the alternatively spliced protein isoforms that are generated in the absence of CELF1, present with a modification of their disordered region, which may be involved in protein-protein interaction. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including. Of these candidates, 10 are found to encode proteins with function related to the cytoskeleton. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Integration of iCLIP and RNA-seq data led to identification of 22 RNAs whose splicing pattern is likely directly controlled by CELF1. This was followed by an integrative analysis with independently generated RNA-seq data on Celf1 cKO and control mouse lenses (omics approach 2) to identify RNAs that are directly bound by CELF1 protein and also exhibit abnormal alternative splicing events resulting from Celf1-deficiency. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) coupled to high-throughput RNA-sequencing (iCLIP-seq) was performed to identify the CELF1 protein binding sites in target RNAs in adult wild-type (WT) mouse lens tissue (omics approach 1). Thus, this study aims to identify CELF1 targets in the lens, and to uncover how the mis-regulated alternatively splice isoforms contribute to cataracts.Īlternatively spliced Celf1-regulated RNA isoforms in the lens were identified by taking two omics approaches. However, a global-level understanding of AS targets of CELF1 in the lens remains unaddressed. These data suggests that Celf1 plays an important role lens formation in vertebrates. Further, it’s knockdown in zebrafish and Xenopus results in lens defects. Celf1 lens conditional deletion in mouse ( Celf1 cKO) results in cataract. Celf1 encodes a conserved RNA-binding protein (RBP) that binds to pre-mRNAs in the nucleus to regulate their alternative splicing (AS).
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