Poster abstracts
Poster number 77 submitted by Cole Lewis
Dual color fluorescent reporters as a tool to study alternative splicing regulation of the Hippo signaling pathway.
Cole Lewis (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Joseph Seimetz (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry and Medical Biochemistry, College of Medicine, Institute of Genomic Biology, University of Illinois, Urbana-Champaign)
Abstract:
The Hippo tumor suppressor pathway is a crucial regulator of cellular proliferation and the maintenance of organ size. We identified a coordinated splicing isoform switch during mouse and human liver development in two crucial genes in the Hippo signaling pathway, NF2 and YAP1. NF2 acts as an upstream regulator while YAP1 is the major downstream effector of the signaling cascade. To define the cis-acting elements and trans-acting factors regulating the NF2 and YAP1 alternative exons, we engineered dual-color fluorescent reporters to monitor their splicing in real time and at single cell resolution. Skipping of the alternative exon within the reporter maintains the reading frame and results in the production of functional dsRED protein, while inclusion results in a frame shift that produces eGFP or BFP. We demonstrate that the fluorescent readouts exhibit strong correlation with the splicing patterns of respective minigenes in a variety of mammalian cell lines. Cotransfection of the reporters with splice site-blocking morpholino oligonucleotides completely abrogates the exon inclusion, demonstrating that the system can be dynamically regulated. Finally, we show that cell confluence has striking effects on the reporter fluorescence output such that rapidly dividing cells at low density produce a mixture of dsRED and eGFP proteins where as contact inhibition due to high cell density results in exclusive production of eGFP. Future efforts will dissect the cell confluence mechanism(s) that dictate the splicing and fluorescence outcome of these reporters.
Keywords: Hippo Pathway, NF2, YAP1