June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Identification of alternative spliced RNA targets of the cataract-linked RNA-binding protein CELF1: use of an multi-omics approach based an iCLIP-seq and RNA-seq
Author Affiliations & Notes
  • Matthieu Duot
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Carole Gautier-Courteille
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Yann Audic
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Agnes Mereau
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Archana Siddam
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Deepti Anand
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • David Reboutier
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Justine Viet
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Catherine Le-goff-gaillard
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Salil Anil Lachke
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Luc Paillard
    Institut de Genetique et Developpement de Rennes, Rennes, Bretagne, France
  • Footnotes
    Commercial Relationships   Matthieu Duot None; Carole Gautier-Courteille None; Yann Audic None; Agnes Mereau None; Archana Siddam None; Deepti Anand None; David Reboutier None; Justine Viet None; Catherine Le-goff-gaillard None; Salil Lachke None; Luc Paillard None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1946. doi:
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      Matthieu Duot, Carole Gautier-Courteille, Yann Audic, Agnes Mereau, Archana Siddam, Deepti Anand, David Reboutier, Justine Viet, Catherine Le-goff-gaillard, Salil Anil Lachke, Luc Paillard; Identification of alternative spliced RNA targets of the cataract-linked RNA-binding protein CELF1: use of an multi-omics approach based an iCLIP-seq and RNA-seq. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1946.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Celf1 encodes a conserved RNA-binding protein (RBP) that binds to pre-mRNAs in the nucleus to regulate their alternative splicing (AS). Celf1 lens conditional deletion in mouse (Celf1cKO) results in cataract. Further, it’s knockdown in zebrafish and Xenopus results in lens defects. These data suggests that Celf1 plays an important role lens formation in vertebrates. However, a global-level understanding of AS targets of CELF1 in the lens remains unaddressed. Thus, this study aims to identify CELF1 targets in the lens, and to uncover how the mis-regulated alternatively splice isoforms contribute to cataracts.

Methods : Alternatively spliced Celf1-regulated RNA isoforms in the lens were identified by taking two omics approaches. 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). This was followed by an integrative analysis with independently generated RNA-seq data on Celf1cKO 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.

Results : Integration of iCLIP and RNA-seq data led to identification of 22 RNAs whose splicing pattern is likely directly controlled by CELF1. Of these candidates, 10 are found to encode proteins with function related to the cytoskeleton. 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. Thus, these data lead to the hypothesis that the abnormal splicing of the cytoskeletal protein-encoding RNAs contributes to cataract in Celf1cKO, by producing protein isoform with an altered interactome.

Conclusions : This study reports the application of two omics approaches to identify new AS targets of Celf1 in the lens. 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 abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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