1. Academic Validation
  2. GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo

GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo

  • Biosci Rep. 2014 Dec 12;34(6):e00163. doi: 10.1042/BSR20140142.
Shengbing Zang 1 Ting-Yu Lin 1 Xinji Chen 1 Marieta Gencheva 1 Alain N S Newo 1 Lixin Yang 1 Daniel Rossi 1 Jianda Hu 2 Shwu-Bin Lin 3 Aimin Huang 4 Ren-Jang Lin 1
Affiliations

Affiliations

  • 1 *Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
  • 2 §Fujian Institute of Hematology, Union Hospital of Fujian Medical University, Fuzhou, China.
  • 3 ‡Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan.
  • 4 †Department of Pathology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
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.

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