September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Identification of PNN-containing nuclear spliceosome and exon junction complexes from HeLa and human corneal epithelial cell
Author Affiliations & Notes
  • Debra Akin
    Anatomy & Cell Biology, University of Florida, Gainesville, Florida, United States
  • Stephen P Sugrue
    Anatomy & Cell Biology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Debra Akin, None; Stephen Sugrue, None
  • Footnotes
    Support  NIH awards R01 EY07883, P30 EY021721
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4375. doi:
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      Debra Akin, Stephen P Sugrue; Identification of PNN-containing nuclear spliceosome and exon junction complexes from HeLa and human corneal epithelial cell. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4375.

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

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Abstract

Purpose : The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Pinin (PNN) has dramatic implications on corneal epithelial differentiation and maintenance of the corneal epithelial cell phenotype. We have shown that lowering of PNN levels in epithelial cells results in dramatic transformation in the amount and composition of splicing variants and that PNN plays a crucial role in the selection of which RNA isoforms differentiating cells produce, implying that PNN exerts broad influence over the regulation and maintenance of epithelial cell phenotype through impacting RNA splicing. Here we report the identification of a PNN complex that contains the essential components for alternative splicing (AS).

Methods : HeLa and human corneal epithelial cells (HCET cells) were engineered to express double-epitope-tagged versions of PNN with bicistronic retroviral vectors pOZFH-C and pOZFH-N. Subsequent to large-scale cell growth and nuclear extraction, the PNN-containing nuclear complexes were isolated by tandem affinity. MS/MS was then utilized for calculation of masses and peptide identifications.

Results : We purified the PNN-containing complex(es) from HeLa cells and analyzed components by mass spectrometry. Multiple core spliceosomal components, such as SNRPD1, SNRPD3, splicing factors, SR proteins, deadbox RNA helicases were found to be complexed with PNN. Interestingly, PNN-complexes also contained a high abundance of exon junction complex (EJC) components SAP18, eIF4AIII, Magoh, Y14 and RNPS1, and Acinus. These data suggest that PNN in associated with both the core spliceosome and the EJC. PNN-containing complexes were then isolated from HCET cells and Western blotting confirmed many of the proteins identified in Hela cells.

Conclusions : These findings identify a molecular mechanism by which PNN mediates modulation of RNA splicing and may influence the epithelial transcriptome. It has been shown that the multiprotein EJC is deposited on mRNAs upstream of exon–exon junctions and serves as a key modulator of spliced mRNA metabolism. We suggest that PNN may influence both RNA splicing and RNA stability through its activity in the spliceosome and EJC.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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