June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Looking for hidden cues: can the function of the moonlighting ocular protein βA3/A1-crystallin be regulated by the signals hidden in its coding sequence?
Author Affiliations & Notes
  • Nadezda A Stepicheva
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Ekaterina Volkova
    Pervyj Moskovskij gosudarstvennyj medicinskij universitet imeni I M Secenova, Moskva, Moskva, Russian Federation
  • Victoria Koontz
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • PENG SHANG
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Haitao Liu
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Sayan Ghosh
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Anastasiia Strizhakova
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Olivia Chowdhury
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Rachel Daley
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • J. Samuel Zigler
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Stacey L Hose
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Debasish Sinha
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Nadezda Stepicheva None; Ekaterina Volkova None; Victoria Koontz None; PENG SHANG None; Haitao Liu None; Sayan Ghosh None; Anastasiia Strizhakova None; Olivia Chowdhury None; Rachel Daley None; J. Zigler None; Stacey Hose None; Debasish Sinha None
  • Footnotes
    Support  This work was supported by the University of Pittsburgh start-up funds, the Jennifer Salvitti Davis, M.D. Chair Professorship in Ophthalmology (DS) and Research to Prevent Blindness (to Ophthalmology, UPMC).
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2300. doi:
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      Nadezda A Stepicheva, Ekaterina Volkova, Victoria Koontz, PENG SHANG, Haitao Liu, Sayan Ghosh, Anastasiia Strizhakova, Olivia Chowdhury, Rachel Daley, J. Samuel Zigler, Stacey L Hose, Debasish Sinha; Looking for hidden cues: can the function of the moonlighting ocular protein βA3/A1-crystallin be regulated by the signals hidden in its coding sequence?. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2300.

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

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Abstract

Purpose : βA3/A1-crystallin (encoded by Cryba1) plays a structural role in the lens but has non-structural functions in the retinal pigment epithelium (RPE). Several isoforms of βA3/A1-crystallin exist, including two peptides (A3 and A1) produced from the same full-length mRNA by leaky ribosomal scanning and a truncated isoform produced from mRNA lacking exon 4. We hypothesize that such isoform plasticity represents a unique mechanism of precise regulation of βA3/A1-crystallin function.

Methods : Phylogenetic analysis of the βA3/A1-crystallin sequence was performed using GeneTree (Ensembl, ENSGT00940000159685). NetPhos3.1 was used for predicting phosphorylation sites. RPE flatmounts and whole lenses (from C57Bl/6J mice) and HPV16 E6/E7 cells were incubated with 100 mg/mL cycloheximide for 3 hours followed by qPCR using Taqman probes designed against different exon junctions of Cryba1 mRNA.

Results : The analysis of the Cryba1 coding sequence (CDS) revealed a CCACC sequence upstream of the second translation initiation site that is used to produce the shorter A1 isoform. This sequence might represent a KOZAK sequence that is “hidden” within the Cryba1 CDS to drive leaky ribosomal scanning. Strikingly, this “hidden” KOZAK translates into threonine residues, which are predicted to be phosphorylated by protein kinase C (PKC, NetPhos score 0.835) and are highly conserved in mammals, except for Marsupials. We also found that the truncated peptide translated from mRNA lacking exon 4 might be produced only to be degraded by nonsense-mediated decay (NMD). Treatment of immortalized mouse RPE cells (but not freshly extracted RPE or lens) with NMD inhibitor cycloheximide, resulted in a reduced proportion of Cryba1 mRNA transcripts that include exon 4, suggesting that NMD may be a primary mechanism involved in shutting down Cryba1 expression as occurs in RPE cell lines.

Conclusions : The finding that the “hidden” KOZAK might not only be responsible for the emerging of a shorter, functionally different A1 isoform but also translates into potentially phosphorylatable threonines may reveal an elegant way to regulate protein function at multiple levels. NMD might offer an additional layer of regulation of Cryba1 expression, however, its mechanism and possible benefits are yet to be studied.

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

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