Talk on Friday 03:15-03:30pm submitted by Cole Lewis
RBFOX2 Controls a Developmental Splicing Switch of the Hippo Pathway Regulator Neurofibromin 2
Cole Lewis (Department of Biochemistry and Medical, University of Illinois, Urbana-Champaign), Waqar Arif (Department of Biochemistry and Medical, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry and Medical, University of Illinois, Urbana-Champaign)
The Hippo signaling pathway is a major regulator of cellular proliferation and organ size. Here we identify an evolutionarily conserved alternative splicing switch in Neurofibromin 2 (NF2), an upstream regulator of the Hippo pathway, during postnatal liver development. Inclusion of NF2 exon 15 (E15) increases in murine hepatocytes after birth and introduces an early stop codon, resulting in an NF2 isoform (NF2-2) with a truncated C-terminal domain (CTD). Three-dimensional structural modeling demonstrated that in contrast to the fetal isoform (NF2-1), NF2-2 is not predicted to form intramolecular interactions between the CTD and the N-terminal FERM domain. As these interactions inhibit NF2’s tumor suppressor activity, NF2-2 is expected to constitutively antagonize cellular proliferation in quiescent adult hepatocytes. To identify the cis-regulatory elements and trans-acting factors controlling this event in hepatocytes, we engineered a dual-color fluorescent reporter to monitor E15 splicing in real-time and at single-cell resolution. Computational analysis of ENCODE eCLIP data from human liver cell line revealed that RBFOX2 binds to a highly conserved region in the intron immediately downstream of E15. Systematic mutational analysis of the cluster of three RBFOX2 binding sites identified a hexamer UGCAUG sequence that drives RBFOX2-mediated NF2 E15 inclusion. Furthermore, using siRNA-knockdown and adenoviral overexpression in mouse and human hepatocytes, we demonstrate that RBFOX2 is both necessary and sufficient for producing the adult NF2-2 splice isoform. Conditional knockout of Rbfox2 gene in mouse livers resulted in near complete skipping of E15, confirming the in vivo requirement for RBFOX2 in maintaining the adult NF2 splicing pattern. Altogether, our data show that by controlling a developmental splicing switch of an upstream regulator of the Hippo signaling pathway, RBFOX2 may serve to fine-tune the proliferation potential of adult hepatocytes.
Keywords: splicing, Hippo signaling pathway