Shuhei Zenno

Short-Interfering-RNA-Mediated Gene Silencing in Mammalian Cells Requires Dicer and eIF2C Translation Initiation Factors

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.

Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target.

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO‐K1) as well, although double‐stranded RNA required for the latter was 2500 times more than for the former. Cognate double‐stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.

Three-dimensional Structure of AzoR from Escherichia coli AN OXIDEREDUCTASE CONSERVED IN MICROORGANISMS

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2Å resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.

Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.

Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin

Aequorin is a photoprotein that emits light in the presence of Ca2+ ions. To develop a bioluminescent immunoassay based on the light emission property of aequorin, we have expressed the apoaequorin fusion protein with S. aureus protein A in E. coli by recombinant DNA techniques. The fusion protein expressed was purified by IgG-Sepharose affinity chromatography, gel filtration and HPLC procedures. The purified protein A-apoaequorin fusion protein has both the luminescent activity of aequorin and the IgG-binding ability of protein A. We compared results obtained using the protein A-aequorin fusion protein with those obtained using a protein A conjugated horseradish peroxidase based immunoassay, and found them to yield similar results.

The Hsp90 Inhibitor Geldanamycin Abrogates Colocalization of eIF4E and eIF4E-Transporter into Stress Granules and Association of eIF4E with eIF4G

The eukaryotic translation initiation factor eIF4E plays a critical role in the control of translation initiation through binding to the mRNA 5′ cap structure. eIF4E is also a component of processing bodies and stress granules, which are two types of cytoplasmic RNA granule in which translationally inactivated mRNAs accumulate. We found that treatment with the Hsp90 inhibitor geldanamycin leads to a substantial reduction in the number of HeLa cells that contain processing bodies. In contrast, stress granules are not disrupted but seem to be only partially affected by the inhibition of Hsp90. However, it is striking that eIF4E as well as its binding partner eIF4E transporter (4E-T), which mediates the import of eIF4E into the nucleus, are obviously lost from stress granules. Furthermore, the amount of eIF4G that is associated with the cap via eIF4E is reduced by geldanamycin treatment. Thus, the chaperone activity of Hsp90 probably contributes to the correct localization of eIF4E and 4E-T to stress granules and also to the interaction between eIF4E and eIF4G, both of which may be needed for eIF4E to acquire the physiological functionality that underlies the mechanism of translation initiation.

Crystal structure of the PIN domain of human telomerase-associated protein EST1A

Saccharomyces cerevisiae Est1p is a telomerase‐associated protein essential for telomere length homeostasis. hEST1A is one of the three human Est1p homologues and is considered to be involved not only in regulation of telomere elongation or capping but also in nonsense‐mediated degradation of RNA. hEST1A is composed of two conserved regions, Est1p homology and PIN (PilT N‐terminus) domains. The present study shows the crystal structure of the PIN domain at 1.8 Å resolution. The overall structure is composed of an α/β fold or a core structure similar to the counterpart of 5′ nucleases and an extended structure absent from archaeal PIN‐domain proteins and 5′ nucleases. The structural properties of the PIN domain indicate its putative active center consisting of invariant acidic amino acid residues, which is geometrically similar to the active center of 5′ nucleases and an archaeal PAE2754 PIN‐domain protein associated with exonuclease activity. Proteins 2007.

Hsp90 is involved in the formation of P-bodies and stress granules

Previously, we found that treatment of cells with the Hsp90 inhibitor geldanamycin (GA) leads to a substantial reduction in the number of processing bodies (P-bodies), and also alters the size and subcellular localization of stress granules. These findings imply that the chaperone activity of Hsp90 is involved in the formation of P-bodies and stress granules. To verify these observations, we examined whether another Hsp90 inhibitor radicicol (RA) affected P-bodies and stress granules. Treatment with RA reduced the level of the Hsp90 client protein Argonaute 2 and the number of P-bodies. Although stress granules still assembled in RA-treated cells upon heat shock, they were smaller and more dispersed in the cytoplasm than those in untreated cells. Furthermore eIF4E and eIF4E-transporter were dissociated selectively from stress granules in RA-treated cells. These observations were comparable to those obtained upon treatment with GA in our previous work. Thus, we conclude that abrogation of the chaperone activity of Hsp90 affects P-body formation and the integrity of stress granules.

Distinguishable In Vitro Binding Mode of Monomeric TRBP and Dimeric PACT with siRNA

RNA interference (RNAi) is an evolutionally conserved posttranscriptional gene-silencing mechanism whereby small interfering RNA (siRNA) triggers sequence-specific cleavage of its cognate mRNA. Dicer, Argonaute (Ago), and either TAR-RNA binding protein (TRBP) or a protein activator of PKR (PACT) are the primary components of the RNAi pathway, and they comprise the core of a complex termed the RNA-induced silencing complex (RISC)-loading complex (RLC). TRBP and PACT share similar structural features including three dsRNA binding domains (dsRBDs), and a complex containing Dicer and either TRBP or PACT is considered to sense thermodynamic asymmetry of siRNA ends for guide strand selection. Thus, both TRBP and PACT are thought to participate in the RNAi pathway in an indistinguishable manner, but the differences in siRNA binding mode and the functional involvement of TRBP and PACT are poorly understood. Here, we show in vitro binding patterns of human TRBP and PACT to siRNA using electrophoresis mobility shift analysis and gel filtration chromatography. Our results clearly showed that TRBP and PACT have distinct in vitro siRNA binding patterns from each other. The results suggest that monomeric TRBP binds to siRNA at the higher affinity compared to the affinity for own homodimerization. In contrast, the affinity between PACT and siRNA is lower than that of homodimerization or that between TRBP and siRNA. Thus, siRNA may be more readily incorporated into RLC, interacting with TRBP (instead of PACT) in vivo.

Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli.

AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to diffract X-rays to beyond 1.8 A resolution using a synchrotron-radiation source. The crystals belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 92.2, c = 51.9 A. The crystals are expected to contain one subunit of the homodimer in the asymmetric unit (VM = 2.6 A3 Da(-1)) and to have a solvent content of 51.6%. Data sets were also collected from heavy-atom derivatives for use in phasing. As a result, crystals soaked in a solution containing K2PtCl4 for 23 d were found to be reasonably isomorphous to the native crystals and the presence of Pt atoms could be confirmed. The data sets from the native crystals and the K2PtCl4-derivatized crystals are being evaluated for use in structure determination by single isomorphous replacement with anomalous scattering.