iPLA 2 β Modulates Bcl-x Splicing 1 Group VIA Phospholipase A 2 (iPLA 2 β) Modulates Bcl-x 5’ Splice Site Selection and Suppresses Anti- Apoptotic Bcl-x(L) in β-cells* Suzanne E. Barbour 1,7 , Phuong T. Nguyen 1,7 , Margaret Park 1 , Bhargavi Emani 1 , Xiaoyong Lei 2 , Mamatha Kambalapalli 1 , Jacqueline C. Shultz 1 , Dayanjan Wijesinghe 1,3 , Charles E. Chalfant 1,3,4,5 , and Sasanka Ramanadham 2, 6 1 Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298-0614 2 Department of Cell, Developmental, and Integrative Biology and Comprehensive Diabetes Center, University of Alabama at Birmingham, AL, 35294 3 Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, Virginia 23249 4 The Massey Cancer Center, Richmond, VA 23298 5 Virginia Commonwealth University Reanimation Engineering Science Center (VCURES), Richmond, Virginia 23298 *Running Title: iPLA 2 β Modulates Bcl-x Splicing in β-cells 6 To Whom Correspondence Should be Addressed: Sasanka Ramanadham, Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, 35294 Tel: 205-966-5973; Fax: 205-966-5220; E-mail: sramvem@uab.edu 7 These individuals contributed equally to the work. Keywords: Group VIA Phospholipase A 2 (iPLA 2 β), Bcl-x(L), β-cell, alternative splicing, diabetes, apoptosis, Bcl-2 family Background: β-cell apoptosis, a critical contributor to T1D, involves iPLA 2 β activation and is suppressed by Bcl-x(L). Results: iPLA 2 β-derived lipids activate an alternative 5’ splice site, reducing protective Bcl- x(L) protein. Conclusion: Modulation of Bcl-x splicing is another key mechanism by which iPLA 2 β-derived lipids promote β-cell apoptosis. Significance: Delineation of molecular mechanisms underlying iPLA 2 β-regulated splicing will elucidate novel strategies to counter β-cell death in T1D. ABSTRACT Diabetes is a consequence of reduced β -cell function and mass, due to β -cell apoptosis. ER stress is induced during β -cell apoptosis due to various stimuli and our work indicates that iPLA 2 β participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces antiapoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(s) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(s) mRNA in INS-1 and islet β -cells. However, chemical inactivation or knockdown of iPLA 2 β http://www.jbc.org/cgi/doi/10.1074/jbc.M115.648956 The latest version is at JBC Papers in Press. Published on March 11, 2015 as Manuscript M115.648956 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on June 18, 2016 http://www.jbc.org/ Downloaded from