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 superfamily. In this study, the functional domains of RH-II/Gu molecule were mapped by fusing the protein or its deletion mutants with a green fluorescence protein and subsequently transfecting or microinjecting the recombinant constructs into HeLa cells. In addition to the identification of a nuclear localization signal (NLS) in the N-terminus and a nucleolar targeting signal 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 domains of the protein in HeLa cells did not interfere with the normal distribution of fibrillarin. However, when the helicase domain was truncated, the distribution pattern of fibrillarin was distorted. Microinjection of the wild-type RH-II/Gu cDNA into the nucleus of HeLa cells did not disrupt normal cell growth. However, when cells were injected with mutant DNA, only a small percentage of HeLa cells progressed through the cell cycle. Analysis of centrosomes in transfected cells demonstrated that most of the mutant-expressing cells were arrested early in the cell cycle. The results suggest that each of the structural domains of RH-II/Gu is necessary for cell growth and cell cycle progression.