title: Bcl-x pre-mRNA splicing regulates brain injury after neonatal hypoxia-ischemia.
abstract: The bcl-x gene appears to play a critical role in regulating apoptosis in the developing and mature CNS and following CNS injury. Two isoforms of Bcl-x are produced as a result of alternative pre-mRNA splicing: Bcl-x(L) (the long form) is anti-apoptotic, while Bcl-x(S) (short form) is pro-apoptotic. Despite the antagonistic activities of these two isoforms, little is known about how regulation of alternative splicing of bcl-x may mediate neural cell apoptosis. Here, we report that apoptotic stimuli (staurosporine or C2-ceramide) reciprocally altered Bcl-x splicing in neural cells, decreasing Bcl-x(L) while increasing Bcl-x(S). Specific knockdown of Bcl-x(S) attenuated apoptosis. To further define regulatory elements that influenced Bcl-x splicing, a Bcl-x minigene was constructed. Deletional analysis revealed several consensus sequences within intron 2 that altered splicing. We found that the splicing factor, CUG-binding-protein-1 (CUGBP1), bound to a consensus sequence close to the Bcl-x(L) 5' splice site, altering the Bcl-x(L)/Bcl-x(S) ratio and influencing cell death. In vivo, neonatal hypoxia-ischemia reciprocally altered Bcl-x pre-mRNA splicing, similar to the in vitro studies. Manipulation of the splice isoforms using viral gene transfer of Bcl-x(S) shRNA into the hippocampus of rats before neonatal hypoxia-ischemia decreased vulnerability to injury. Moreover, alterations in nuclear CUGBP1 preceded Bcl-x splicing changes. These results suggest that alternative pre-mRNA splicing may be an important regulatory mechanism for cell death after acute neurological injury and may potentially provide novel targets for intervention.
authors.affiliation: Hope Center for Neurological Disorders and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Qingli
authors.initials: Q
authors.lastname: Xiao
authors.forename: Andria L
authors.initials: AL
authors.lastname: Ford
authors.forename: Jan
authors.initials: J
authors.lastname: Xu
authors.forename: Ping
authors.initials: P
authors.lastname: Yan
authors.forename: Kuang-Yung
authors.initials: KY
authors.lastname: Lee
authors.forename: Ernesto
authors.initials: E
authors.lastname: Gonzales
authors.forename: Tim
authors.initials: T
authors.lastname: West
authors.forename: David M
authors.initials: DM
authors.lastname: Holtzman
authors.forename: Jin-Moo
authors.initials: JM
authors.lastname: Lee
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001930:Brain Injuries / Q000209:etiology*; D000067879:CELF1 Protein; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007839:Functional Laterality; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D020925:Hypoxia-Ischemia, Brain / Q000150:complications*; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D011247:Pregnancy; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000067879:CELF1 Protein; C000594460:Celf1 protein, rat; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D012322:RNA Precursors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D051020:bcl-X Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase; D019311:Staurosporine; D013110:Sphingosine