An integrative analysis using iCLIP-seq and RNA-Seq to identify genes post-transcriptionally regulated by the cataract-linked RNA-binding protein CELF1 in the lens

Abstract

Purpose : Celf1 encodes an RNA-binding protein (RBP) that post-transcriptionally regulates gene expression by distinct mechanisms, including alternative splicing. Germline knockout (KO) or lens-specific conditional knockout (cKO) Celf1 mice develop lens defects and early-onset cataract. We sought to identify RNA targets of CELF1 protein in the lens, particularly those regulated at the level of alternative splicing, and whose mis-regulation upon CELF1-deficiency may contribute to lens defects and cataract.

Methods : We performed integrative analysis using multiple omics-based approaches to identify Celf1-regulated downstream RNA targets in the lens. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) followed by high-throughput RNA-sequencing (iCLIP-seq) identified CELF1 protein-RNA binding sites in adult mouse wild-type (WT) lens. This data was analyzed in the context of RNA-seq data on Celf1 KO or Celf1 cKO and control mouse lenses, which identified mis-regulated candidate genes with alternative splicing events.

Results : Integrative analysis of iCLIP-seq and RNA-seq data allowed the prioritization of 22 RNAs whose splicing pattern is likely directly controlled by CELF1 protein in the lens. Of these, seven RNAs that encode proteins linked to cytoskeleton (e.g. ABLIM1, ANK2, CLTA, CTNNA2, SPTBN1, SEPTIN8, YWHAE) were validated by RT-PCR to have mis-expressed alternative splice isoforms in Celf1 KO lens. Normal lens fiber cell differentiation involves cytoskeleton modifications that are necessary for their characteristic morphology and organization, which is found to be perturbed in Celf1-deficient lens. Thus, abnormal splicing of these cytoskeletal protein-encoding RNAs may contribute to the cataract pathology in Celf1 KO lenses.

Conclusions : Integrative analysis using iCLIP-seq data generated on WT lens (for identifying RNA binding sites of CELF1 protein) and RNA-seq data generated on Celf1 KO and Celf1 cKO lens (for identifying genes with mis-regulated alternative splicing event) led to prioritization of targets that are linked to the cytoskeleton and whose altered expression may explain distinct aspects of the lens pathology and cataract resulting from Celf1 deficiency.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.