Celf1 regulates the small heat shock protein Hspb8 in lens development

Abstract

Purpose : Small heat shock proteins (sHSPs) are involved in the maintenance of cellular homeostasis under stress conditions. Several sHSPs containing an alpha crystallin domain are expressed in the mouse lens. However, our understanding of expression control of sHSPs, especially on the post-transcriptional level, is limited. We address this knowledge-gap by examining the regulatory relationship between a cataract-linked RNA-binding protein (RBP), Celf1, and the sHSP, Hspb8, in mouse lens development.

Methods : Celf1 compound conditional lens-specific deletion mice were generated by using Pax6GFPCre, Celf1 flox and germline knock-in alleles (termed Celf1^cKO/lacZKI). Genome-level expression profiling on Celf1^cKO/lacZKI mouse lenses at stage P0 was performed by microarray on the Illumina MouseWG-6 v2.0 BeadChip platform and RNA-sequencing (RNA-seq). Celf1^cKO/lacZKI mouse lenses at stage P0 were also analyzed by Tandem mass tag (TMT) labelled quantitative proteomics. Immunofluorescence (IF) was performed on lens sections using Hspb8 antibody (Abcam). Celf1 RNA-IP (RIP) was performed using RIP-kit (Millipore) on post-natal day (P) 15 wild-type mouse lens lysates followed by RT-qPCR. Wild-type P13 mouse lenses were UV cross-linked and subjected to Celf1-IP for CLIP assays.

Results : Two transcriptome screens (microarray, RNA-seq) as well as a proteome-based screen independently identified a key heat-shock factor, Hspb8, to be abnormally overexpressed at both mRNA and protein levels in Celf1^cKO/lacZKI lenses. RT-qPCR and IF analysis of Celf1-deficient lenses validate Hspb8 overexpression. RIP and CLIP assays demonstrate that Celf1 protein directly binds Hspb8 mRNA. These data suggests that Celf1 may mediate post-transcriptional to negatively control Hspb8 expression in lens development. Because Hspb8 is involved in multiple cellular processes ranging from cytoskeletal maintenance to repairing damaged proteins, these data provide new insights into its expression control.

Conclusions : Hspb8 harbors a conserved alpha-crystallin domain and functions as a molecular chaperone, binding partially unfolded polypeptides to maintain them in a refolding-competent state. This study suggests that Celf1 negatively controls Hspb8 expression in the lens.

This is a 2020 ARVO Annual Meeting abstract.