Satoshi Shibata

A high-resolution structure of the pre-microRNA nuclear export machinery

Pre-MicroRNA Export Machinery Micro (mi) RNAs play a role in the regulation of many biological processes. Long transcripts are initially processed in the nucleus to yield pre-miRNAs that are translocated through the nuclear pore complex and further processed to mature miRNAs in the cytoplasm. Okada et al. (p. 1275; see the Perspective by Stewart) describe the crystal structure of pre-miRNA complexed with the exportin Exp5 and the small nuclear GTPase RanGTP. The structure shows that Exp5 and RanGTP protect the miRNA from degradation by nucleases, as well as facilitate transport to the cytoplasm. RNA recognition is mainly through ionic interactions that are sequence independent, and model-building suggests that this nuclear export machinery could accommodate other small-structured RNAs. Exportin-5:RanGTP surrounds microRNAs to protect them from degradation as it exports them from the nucleus. Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)–bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3′ overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt–like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3′ overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.

Cellular stresses induce the nuclear accumulation of importin α and cause a conventional nuclear import block

We report here that importin α accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin α appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin α. In addition, nuclear retention and the importin β/Ran-independent import of importin α also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin α from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin α is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin α can be involved in a common physiological response to various stress conditions.

Exportin-5 orthologues are functionally divergent among species

Exportin-5, an evolutionarily conserved nuclear export factor belonging to the importin-β family of proteins, is known to play a role in the nuclear export of small noncoding RNAs such as precursors of microRNA, viral minihelix RNA and a subset of tRNAs in mammalian cells. In this study, we show that the exportin-5 orthologues from different species such as human, fruit fly and yeast exhibit diverged functions. We found that Msn5p, a yeast exportin-5 orthologue, binds double-stranded RNAs and that it prefers a shorter 22 nt, double-stranded RNA to ∼80 nt pre-miRNA, even though both of these RNAs share a similar terminal structure. Furthermore, we found that Drosophila exportin-5 binds pre-miRNAs and that amongst the exportin-5 orthologues tested, it shows the highest affinity for tRNAs. The knockdown of Drosophila exportin-5 in cultured cells decreased the amounts of tRNA as well as miRNA, whereas the knock down of human exportin-5 in cultured cells affected only miRNA but not tRNA levels. These results indicate that double-stranded RNA binding ability is an inherited functional characteristic of the exportin-5 orthologues and that Drosophila exportin-5 functions as an exporter of tRNAs as well as pre-miRNAs in the fruit fly that lacks the orthologous gene for exportin-t.

Nucleocytoplasmic transport of proteins and poly(A)+ RNA in reconstituted Tpr-less nuclei in living mammalian cells.

Background: It is known that Tpr is a component of an intranuclear long filament which extends from the nuclear pore complex (NPC) into the nucleoplasm. Since the over‐expression of the full‐length of or some fragments of Tpr in living cells leads to the accumulation of poly(A)+ RNA within the nuclei, it is generally thought that a relationship exists between Tpr and the nuclear export of mRNA in mammalian cells. In contrast, the nuclear export of poly(A)+ RNA was not inhibited in a double deletion mutant of yeast Tpr homologues (Mlp1p and Mlp2p). Therefore, the precise function of Tpr remains unknown.

Direct Synthesis of Hydrogen Peroxide from Hydrogen and Oxygen by Using a Water‐Soluble Iridium Complex and Flavin Mononucleotide

H2 , O2 to H2 O2 : The direct synthesis of hydrogen peroxide from hydrogen and oxygen in water has been made possible by using an iridium(III) complex, [Ir(III) (Cp*)(4-(1H-pyrazol-1-yl-κN(2) )benzoic acid-κC(3) )(H2 O)]2 SO4 , and flavin mononucleotide. This method gives hydrogen peroxide with a high turnover number (847) and yield (19.2 %) under normal pressure and at room temperature.

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1–332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

Identification of Ewing's sarcoma gene product as a glycoprotein using a monoclonal antibody that recognizes an immunodeterminant containing O-linked n-acetylglucosamine moiety

The Ewings sarcoma (EWS) oncogene is fused to a variety of cellular transcription factors in various forms of human cancers. Although EWS fusion proteins have been extensively studied, the normal function of EWS remains poorly characterized. We previously reported that a monoclonal antibody, referred to as MY95, recognized nucleoporins such as p62, Nup98, and CAN/Nup214 and an uncharacterized polypeptide with an apparent molecular mass of 83 kDa. In the present study, an amino acid sequence analysis of this 83-kDa protein revealed that it is, in fact, EWS, which is not known to belong to the nucleoporins. We further demonstrated that the immunodeterminant of MY95 contains an N-acetylglucosamine moiety, indicating that EWS is a glycoprotein. Interestingly, the glycosylation level of EWS changes during the neural differentiation of P19 cells. MY95 will be quite useful in further studies of the glycosylated form of EWS in terms of understanding the normal cellular function of this oncogene product.

A chromodomain-containing nuclear protein, MRG15 is expressed as a novel type of dendritic mRNA in neurons.

On the basis of a hypothesis that proteins encoded by the mRNAs that are transported to and translated at the dendrites/synapses may play key roles in synaptic plasticity, this study reports on attempts to isolate mRNAs which are localizing at the dendrites/synapses from mouse cerebellar synaptosomal fractions. Among 100 pieces of dendritic mRNA candidates, 10 pieces of mRNAs were found to contain the cytoplasmic polyadenylation element (CPE)-like sequences which were contained in certain mRNAs translated in dendrites. We next examined the issue of whether the CPE-like sequence-containing mRNAs (CPERs) were localized in the synapses/dendrites by means of in situ hybridization. The findings indicate that CPER9 was actually localized at the apical dendrites of a portion of cerebral cortex layer V pyramidal cells, as well as at the proximal dendrites of some of the cerebellar Purkinje cells. CPER9 was found to encode a mouse homolog of MRG15, a nuclear protein which contains a chromodomain identified in several proteins that act as regulators of transcription. Immunohistochemistry with anti-MRG15 antibodies revealed that MRG15 was localized in dendrites as well as in the nuclei of Purkinje cells. These results suggest that MRG15 may serve as a link between synaptic activity and gene expression.

Catalytic Formation of Hydrogen Peroxide from Coenzyme NADH and Dioxygen with a Water-Soluble Iridium Complex and a Ubiquinone Coenzyme Analogue.

A ubiquinone coenzyme analogue (Q0: 2,3-dimethoxy-5-methyl-1,4-benzoquinone) was reduced by coenzyme NADH to yield the corresponding reduced form of Q0 (Q0H2) in the presence of a catalytic amount of a [C,N] cyclometalated organoiridium complex (1: [Ir(III)(Cp*)(4-(1H-pyrazol-1-yl-κN(2))benzoic acid-κC(3))(H2O)]2SO4) in water at ambient temperature as observed in the respiratory chain complex I (Complex I). In the catalytic cycle, the reduction of 1 by NADH produces the corresponding iridium hydride complex that in turn reduces Q0 to produce Q0H2. Q0H2 reduced dioxygen to yield hydrogen peroxide (H2O2) under slightly basic conditions. Catalytic generation of H2O2 was made possible in the reaction of O2 with NADH as the functional expression of NADH oxidase in white blood cells utilizing the redox cycle of Q0 as well as 1 for the first time in a nonenzymatic homogeneous reaction system.

Catalytic oxidation of formic acid by dioxygen with an organoiridium complex

Catalytic oxidation of formic acid by dioxygen occurred efficiently using an organoiridium complex ([IrIII(Cp*)(4-(1H-pyrazol-1-yl-κN2)benzoic acid-κC3)(H2O)]2SO4, 1) as a catalyst in a water-containing organic solvent as well as in water at ambient temperature. The catalytic cycle is composed of the reduction of 1 by formate to produce the hydride complex, which reduces dioxygen to water to regenerate 1.