Zang Shengbing, Lin Ting-Yu, Chen Xinji, Gencheva Marieta, Newo Alain N S, Yang Lixin, Rossi Daniel, Hu Jianda, Lin Shwu-Bin, Huang Aimin, Lin Ren-Jang
*Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California, U.S.A.
§Fujian Institute of Hematology, Union Hospital of Fujian Medical University, Fuzhou, China.
Biosci Rep. 2014 Dec 12;34(6):e00163. doi: 10.1042/BSR20140142.
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.
人类GPKOW [G-补丁（富含甘氨酸）结构域和KOW（基尔皮德斯、乌佐尼斯和沃斯）结构域] 蛋白包含一个G-补丁结构域和两个KOW结构域，是拟南芥MOS2和酿酒酵母Spp2蛋白的同源物。GPKOW存在于人类剪接体中，但其在mRNA前体剪接中的作用仍有待阐明。在本报告中，我们表明GPKOW直接与DHX16/hPRP2以及RNA相互作用。从HeLa细胞核提取物中免疫去除GPKOW会导致产生一种仍与DHX16结合但无活性的剪接体。添加重组GPKOW可使耗尽提取物恢复剪接活性。在体内，GPKOW的过表达部分抑制了在表达显性负性DHX16突变体的细胞中观察到的剪接缺陷。G-补丁结构域的突变极大地减弱了GPKOW与DHX16的相互作用；然而，该突变体在剪接中具有活性，并且能够抑制剪接缺陷。KOW1结构域的突变略微改变了GPKOW与RNA的相互作用，但该突变体在体外和体内的功能较弱。我们的结果表明，GPKOW在功能上可影响DHX16，但该活性并不需要这两种蛋白之间的相互作用。
