title: GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo.
abstract: Human GPKOW [G-patch (glycine-rich) domain and KOW (Kyrpides, Ouzounis and Woese) domain] protein contains a G-patch domain and two KOW domains, and is a homologue of Arabidopsis MOS2 and Saccharomyces Spp2 protein. GPKOW is found in the human spliceosome, but its role in pre-mRNA splicing remains to be elucidated. In this report, we showed that GPKOW interacted directly with the DHX16/hPRP2 and with RNA. Immuno-depletion of GPKOW from HeLa nuclear extracts resulted in an inactive spliceosome that still bound DHX16. Adding back recombinant GPKOW restored splicing to the depleted extract. In vivo, overexpression of GPKOW partially suppressed the splicing defect observed in dominant-negative DHX16 mutant expressing cells. Mutations at the G-patch domain greatly diminished the GPKOW-DHX16 interaction; however, the mutant was active in splicing and was able to suppress splicing defect. Mutations at the KOW1 domain slightly altered the GPKOW-RNA interaction, but the mutant was less functional in vitro and in vivo. Our results indicated that GPKOW can functionally impact DHX16 but that interaction between the proteins is not required for this activity.
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Shengbing
authors.initials: S
authors.lastname: Zang
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Lin
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Xinji
authors.initials: X
authors.lastname: Chen
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Marieta
authors.initials: M
authors.lastname: Gencheva
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Alain N S
authors.initials: AN
authors.lastname: Newo
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Lixin
authors.initials: L
authors.lastname: Yang
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rossi
authors.affiliation: §Fujian Institute of Hematology, Union Hospital of Fujian Medical University, Fuzhou, China.
authors.forename: Jianda
authors.initials: J
authors.lastname: Hu
authors.affiliation: ‡Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan.
authors.forename: Shwu-Bin
authors.initials: SB
authors.lastname: Lin
authors.affiliation: †Department of Pathology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
authors.forename: Aimin
authors.initials: A
authors.lastname: Huang
authors.affiliation: *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
authors.forename: Ren-Jang
authors.initials: RJ
authors.lastname: Lin
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D024202:Electrophoretic Mobility Shift Assay; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: C000600438:GPKOW protein, human; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012313:RNA; C555647:DHX16 protein, human; D020365:RNA Helicases