Mapping and characterization of the functional domains of the nucleolar protein RNA helicase II/Gu

RNA helicase II/Gu (RH-II/Gu) is a nucleolar RNA helicase of the DEAD-box s uperfamily. In this study, the functional domains of RH-II/Gu molecule were mapped by fusing the protein or its deletion mutants with a green fluoresc ence protein and subsequently transfecting or microinjecting the recombinan t constructs into HeLa cells. In addition to the identification of a nuclea r localization signal (NLS) in the N-terminus and a nucleolar targeting sig nal in the central helicase domain, a hidden NLS and a nucleolar targeting signal were found in the C-terminal arginine/glycine-rich domain. RH-II/Gu colocalized with fibrillarin, a component of the dense fibrillar region of the nucleolus. Overexpression of the entire RH-II/Gu protein or specific do mains of the protein in HeLa cells did not interfere with the normal distri bution of fibrillarin. However, when the helicase domain was truncated, the distribution pattern of fibrillarin was distorted. Microinjection of the w ild-type RH-II/Gu cDNA into the nucleus of HeLa cells did not disrupt norma l cell growth. However, when cells were injected with mutant DNA, only a sm all percentage of HeLa cells progressed through the cell cycle. Analysis of centrosomes in transfected cells demonstrated that most of the mutant-expr essing cells were arrested early in the cell cycle. The results suggest tha t each of the structural domains of RH-II/Gu is necessary for cell growth a nd cell cycle progression. (C) 1999 Academic Press.