June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Deficiency of the transcription elongation factor Ell2, a downstream target of the cataract-linked RNA-binding protein Celf1, causes eye defects
Author Affiliations & Notes
  • Sanjaya Shrestha
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Sandeep Aryal
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Archana Siddam
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Francisco Hernandez
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Salil Anil Lachke
    Department of Biological Sciences, University of Delaware, Newark, Delaware, United States
  • Footnotes
    Commercial Relationships   Sanjaya Shrestha None; Sandeep Aryal None; Archana Siddam None; Francisco Hernandez None; Salil Lachke None
  • Footnotes
    Support  NIH/NEI R01 EY021505
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 653 – F0008. doi:
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      Sanjaya Shrestha, Sandeep Aryal, Archana Siddam, Francisco Hernandez, Salil Anil Lachke; Deficiency of the transcription elongation factor Ell2, a downstream target of the cataract-linked RNA-binding protein Celf1, causes eye defects. Invest. Ophthalmol. Vis. Sci. 2022;63(7):653 – F0008.

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

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Abstract

Purpose : We recently showed that the cataract-associated RNA-binding protein (RBP) Celf1 post-transcriptionally controls the key transcription factors Pax6 and Prox1 in the lens. To further understand the regulatory relationship between RBPs and the factors that control transcription in the lens, we focused on Ell2 (Elongation factor for RNA Polymerase II 2), which we found to be abnormally over-expressed in the lens in Celf1 lens-specific conditional knockout (Celf1cKO) mice and which was independently identified by iSyTE as a high-priority candidate gene in the lens.

Methods : Ell2 expression in lens development was evaluated by RT-PCR, in situ hybridization (ISH), Western blotting (WB) and immunostaining (IF). Germline (Ell2-/- ) and lens-specific conditional KO mice (Ell2cKO ) were generated by crossing mice carrying Ell2 floxed alleles with CMVCre and Pax6GFPCre mouse lines, respectively.

Results : Ell2 overexpression in Celf1cKO mouse lens as suggested by microarrays and RNA-sequencing was independently validated by RT-qPCR. Further, RNA-immunoprecipitation (RIP) using Celf1 antibody indicated that Celf1 protein directly binds Ell2 RNA in lens cells. These data, along with iSyTE-based lens-enriched expression analysis, identified Ell2 to be a high-priority target in the lens. RT-PCR confirmed robust Ell2 RNA expression in mouse lens at various stages and ISH showed that Ell2 RNA is present in fiber cells near the transition zone at E16.5. WB confirmed Ell2 protein (~72 kDa) in mouse lens and immunostaining showed Ell2 protein levels in lens fiber cell nuclei at embryonic and postnatal stages. In agreement with Ell2’s robust expression in the lens, Ell2-/- and Ell2cKO mice showed ocular defects, including small eye and lens abnormalities.

Conclusions : Ell2 has been shown to interact with RNA polymerase II and increase its elongation rate along template DNA by relieving the transient “pause” in the early stages of transcription. While Ell2 has been associated with cancer and other defects, its role in eye development has not been examined. This study identifies Ell2 as a novel downstream target of the cataract-linked gene Celf1 and validates its robust expression at the RNA and protein levels in the lens. Further, Ell2-/- and Ell2cKO mice exhibit ocular defects indicating its important role in the eye and lens.

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

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