Identification of toposome, a novel multisubunit complex containing topoisomerase IIalpha

Topoisomerase IIalpha plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIalpha exerts its function during mitosis. In this report, we find that topoisomerase IIalpha forms a multisubunit complex, named toposome, containing two ATPase/helicase proteins (RNA helicase A and RHII/Gu), one serine/threonine protein kinase (SRPK1), one HMG protein (SSRP1), and two pre-mRNA splicing factors (PRP8 and hnRNP C). Toposome separates entangled circular chromatin DNA about fourfold more efficiently than topoisomerase IIalpha. Interestingly, this decatenation reaction yields knotted circles, which are not seen in reactions provided with monomeric circular DNA. Our results also show that interaction among toposome-associated proteins is highest in G2/M phase but drastically diminishes in G1/S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.     

Expression,cellular localization,and enzymatic activities of RNA helicase II/Gu(beta)

RNA helicase II/Gu (RH-II/Gu) is a nucleolar DEAD-box protein that unwinds double-stranded RNA and introduces secondary structure to a single-stranded RNA. We recently identified its paralogue, RH-II/Gu(beta), in contrast to the original RH-II/Gu(alpha). Their similar intron-exon structures on chromosome 10 suggest gene duplication. To determine functional differences, their expression, localization, and enzymatic activities were compared. RH-II/Gu(alpha) is expressed two- to threefold more than RH-II/Gu(beta) in most tissues. Both proteins localize to nucleoli, suggesting roles in ribosomal RNA production, but RH-II/Gu(beta) also localizes to nuclear speckles containing splicing factor SC35, suggesting possible involvement in pre-mRNA splicing. The C-terminus responsible for nuclear speckle localization of RH-II/Gu(beta) contains an arginine-serine-rich domain present in some RNA splicing proteins. In vitro assays show weaker ATPase and RNA helicase activities of RH-II/Gu(beta). RH-II/Gu(alpha) unwinds RNA substrate with a 21- or 34-nt duplex and 5' overhangs, but RH-II/Gu(beta) unwinds only the shorter duplex. Although RH-II/Gu(beta) has no RNA folding activity, it catalyzes formation of an RNA complex with unidentified structure, which is not observed when assayed with a mixture of the two enzymes. Instead, the presence of RH-II/Gu(beta) stimulates RH-II/Gu(alpha) unwinding activity. Our data suggest distinct and complex regulation of expression of the two paralogues with nonredundant gene products.     

Functional interaction between RNA helicase II/Gu(alpha) and ribosomal protein L4

RNA helicase II/Gu(alpha) is a multifunctional nucleolar protein involved in ribosomal RNA processing in Xenopus laevis oocytes and mammalian cells. Downregulation of Gu(alpha) using small interfering RNA (siRNA) in HeLa cells resulted in 80% inhibition of both 18S and 28S rRNA production. The mechanisms underlying this effect remain unclear. Here we show that in mammalian cells, Gu(alpha) physically interacts with ribosomal protein L4 (RPL4), a component of 60S ribosome large subunit. The ATPase activity of Gu(alpha) is important for this interaction and is also necessary for the function of Gu(alpha) in the production of both 18S and 28S rRNAs. Knocking down RPL4 expression using siRNA in mouse LAP3 cells inhibits the production of 47/45S, 32S, 28S, and 18S rRNAs. This inhibition is reversed by exogenous expression of wild-type human RPL4 protein but not the mutant form lacking Gu(alpha)-interacting motif. These observations have suggested that the function of Gu(alpha) in rRNA processing is at least partially dependent on its ability to interact with RPL4.     

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.     

Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein.

RNA helicase II/Gu (RH II/Gu) is a nucleolar protein that unwinds dsRNA in a 5' to 3' direction, and introduces a secondary structure into a ssRNA. The helicase domain is at the N-terminal three-quarters of the molecule and the foldase domain is at the C-terminal quarter. The RNA folding activity of RH II/Gu is not a mere artifact of its binding to RNA. This study narrows down the RNA foldase domain to amino acids 749-801 at the C-terminus of the protein. Dissection of this region by deletion and site-directed mutagenesis shows that the four FRGQR repeats, as well as the C-terminal end bind RNA independently. These juxtaposed subdomains are both important for the RNA foldase activity of RH II/Gu. Mutation of either repeat 2 or repeat 4, or simultaneous mutation of Lys792, Arg793 and Lys797 at the C-terminal end of RH II/Gu to alanines inhibits RNA foldase activity. The last 17 amino acids of RH II/Gu can be replaced by an RNA binding motif from nucleolar protein p120 without a deleterious effect on its foldase activity. A model is proposed to explain how RH II/Gu binds and folds an RNA substrate.     

Solution structure of the GUCT domain from human RNA helicase II/Gu beta reveals the RRM fold, but implausible RNA interactions

Human RNA helicase II/Gu alpha (RH-II/Gu alpha) and RNA helicase II/Gu beta (RH-II/Gu beta) are paralogues that share the same domain structure, consisting of the DEAD box helicase domain (DEAD), the helicase conserved C-terminal domain (helicase_C), and the GUCT domain. The N-terminal regions of the RH-II/Gu proteins, including the DEAD domain and the helicase_C domain, unwind double-stranded RNAs. The C-terminal tail of RH-II/Gu alpha, which follows the GUCT domain, folds a single RNA strand, while that of RH-II/Gu beta does not, and the GUCT domain is not essential for either the RNA helicase or foldase activity. Thus, little is known about the GUCT domain. In this study, we have determined the solution structure of the RH-II/Gu beta GUCT domain. Structural calculations using NOE-based distance restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with beta-alpha-alpha-beta-beta-alpha-beta topology in the region for K585-A659, while the Pfam HMM algorithm defined the GUCT domain as G571-E666. This structure-based domain boundary revealed false positives in the sequence homologue search using the HMM definition. A structural homology search revealed that the GUCT domain has the RRM fold, which is typically found in RNA-interacting proteins. However, it lacks the surface-exposed aromatic residues and basic residues on the beta-sheet that are important for the RNA recognition in the canonical RRM domains. In addition, the overall surface of the GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around the surface-exposed tryptophan.