Caspase-mediated processing of TRBP regulates apoptosis during viral infection.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells.

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5' proximal nucleotides, which include seed sequence (positions 2-8 from the 5' end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19-21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15-18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.

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.

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.

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.

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.

Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2-8 from the 5'end of the guide strand; its complementary sequence; the 5'end of the guide strand and the 3'overhang of the passenger strand. However, most part of the 3' two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3'end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA-RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA-RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.

Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer.

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 A resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3' overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 A, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.

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 A resolution. The overall structure is composed of an alpha/beta 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.

Crystallization and preliminary X-ray analysis of the PIN domain of human EST1A.

Human EST1A (ever shorter telomeres 1A) is associated with most or all active telomerase in cell extracts and is involved either directly or indirectly in telomere elongation and telomere capping. The C-terminal region of EST1A contains the PIN (PilT N-terminus) domain, a putative nuclease domain. The PIN domain of human EST1A was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 107.3, b = 51.6, c = 100.5 angstroms, beta = 119.3 degrees, and diffracted X-rays to 1.8 angstroms resolution. The asymmetric unit contained two molecules of the PIN domain and the solvent content was 57%.

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 2.2 A 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.

Crystallization and preliminary X-ray analysis of the C-terminal RNase III domain of human Dicer.

Human Dicer protein contains two RNase III domains (RNase IIIa and RNase IIIb) which are involved in the production of short interfering RNAs (siRNAs). The C-terminal RNase III domain (RNase IIIb) of human Dicer was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C222(1), with unit-cell parameters a = 88.6, b = 199.7, c = 119.6 angstroms, and diffracted X-rays to 2.0 angstroms resolution. The asymmetric unit contained three molecules of the RNase IIIb and the solvent content was 67%.

RNAi induced in mammalian and Drosophila cells via transfection of dimers and trimers of small interfering RNA.

Twenty one base pair long small interfering RNAs (siRNAs) are widely in use in mammalian RNAi experiments. The present study assesses the capability of 43 and 63bp dsRNAs with two 2nt long 3'-overhangs to induce RNAi in mammalian and Drosophila cells. Human Dicer was found to cleave these dsRNAs from their ends to generate two or three monomeric siRNA units, each 21-22bp in length. When, in 43bp dsRNA, there was present a highly-effective siRNA sequence in frame with respect to the Dicer digestion, considerably high RNAi activity was noted to be induced in mouse embryonic stem E14TG2a, human HeLa, Chinese hamster CHO-K1 or Drosophila S2 cells. In contrast, RNAi depending on 63bp dsRNA, containing a highly effective siRNA sequence in frame with respect to Dicer digestion, varied considerably depending on cell lines used. While there was no appreciable RNAi in HeLa cells associated with relatively strong interferon response, a significant level of RNAi was noted in E14TG2a, CHO-K1 and S2 cells, in all of which interferon response induction was but slight, if at all. It would thus follow that siRNA oligomers with sequence of a highly functional siRNA monomer unit in frame with respect to dicer digestion should serve as a good RNAi agent in Drosophila and certain mammalian cells.

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.

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.

Ferulic acid production from clove oil by Pseudomonas fluorescens E118.

For the production of the useful antioxidant ferulic acid from clove oil containing abundant eugenol, the growth of eugenol-degrading microorganisms in the presence of clove oil was examined. Pseudomonas fluorescens E118, a clove-oil-tolerant strain, accumulated 6.1 g/l ferulic acid under optimized culture conditions with the intermittent addition of eugenol. When the bacterium was applied to ferulic acid production from clove oil, 5.8 g/l ferulic acid was produced with the intermittent addition of clove oil. Since clove oil is much cheaper than eugenol, ferulic acid production from clove oil by the bacterium is promising for the industrial production of ferulic acid.