Yasuhito Ishigaki

Evidence for a Pioneer Round of mRNA Translation: mRNAs Subject to Nonsense-Mediated Decay in Mammalian Cells Are Bound by CBP80 and CBP20

Nonsense-mediated decay (NMD) eliminates mRNAs that prematurely terminate translation. We used antibody to the nuclear cap binding protein CBP80 or its cytoplasmic counterpart eIF4E to immunopurify RNP containing nonsense-free or nonsense-containing transcripts. Data indicate that NMD takes place in association with CBP80. We defined other components of NMD-susceptible mRNP as CBP20, PABP2, eIF4G, and the NMD factors Upf2 and Upf3. Consistent with the dependence of NMD on translation, the NMD of CBP80-bound mRNA is blocked by cycloheximide or suppressor tRNA. These findings provide evidence that translation can take place in association with CBP80. They also indicate that CBP80-bound mRNA undergoes a pioneer round of translation, before CBP80-CBP20 are replaced by eIF4E, and Upf2 and Upf3 proteins dissociate from upstream of exon-exon junctions.

The exon junction complex is detected on CBP80‐bound but not eIF4E‐bound mRNA in mammalian cells: dynamics of mRNP remodeling

Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon–exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense‐mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap‐binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap‐binding protein eIF4E. These and other data indicate that NMD targets CBP80‐bound mRNA during a ‘pioneer’ round of translation, but whether nuclear eIF4E also binds nascent but dead‐end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron‐containing RNA and the C‐terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80‐bound but not eIF4E‐bound mRNA. Each of these proteins is also detected on CBP80‐bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.

Photodegradation of 4-alkylphenols using BiVO4 photocatalyst under irradiation with visible light from a solar simulator

Abstract A visible-light-driven photocatalyst, BiVO 4 , has been applied for degradation of a series of linear 4- n -alkylphenols under irradiation from a solar simulator. Degradation rates become faster with increasing alkyl chain length. Half-life of 4- n -nonylphenol (4- n -NP) is 18xa0min, which is about eight times shorter than that of phenol. The rate law of disappearance for heptyl-, octyl- and nonylphenols exhibits zero-order kinetics. The amount of adsorption on BiVO 4 surface is much larger for longer hydrophobic alkylphenols. It is thus concluded that BiVO 4 is suitable for degradation of hydrophobic alkylphenols such as nonyl- and octylphenols nominated as endocrine disruptors. Degradation of branched 4-nonylphenol (mixture of isomers) has also been examined, and disappearance of its estrogenic activity was confirmed by means of the yeast two-hybrid assay. A gas chromatography–mass spectroscopy (GC–MS) analysis after solid-phase extraction indicates that the major product is cis , cis -4-alkyl-6-oxo-2,6-hexadienoic acids, and the minor products are 4-alkylcatechols and 4-(1-alkenyl)phenols.

Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway.

Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells. Here, we demonstrate that the estimated 3 x 10(6) copies of hUpf1 p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively.

Development and characterization of a DNA solar dosimeter.

In this paper, we report the development and characterization of a solar ultraviolet (UV) dosimetry system that can be used as a film badge for radiation monitoring. DNA molecules are coated on a thin nylon membrane as a UV dosimeter. The membrane is sealed in a polyethylene filter envelope with silica gel to keep the humidity low. After exposure to UV or solar light, induced DNA damage is measured by an immunochemical reaction. The intensity of color developed during the immunological reaction can be correlated linearly with the irradiated UV dose delivered by an Oriel solar simulator within a limited dose range. We observe no effects of temperature on the level of damage induction. The membrane is proficient for measuring DNA damage for more than 21 days when stored at either 37 or 4 degrees C. The induced damage remains stable on the membrane for at least 22 days at both 37 and 4 degrees C. In addition to these indoor experiments, we report measurements of solar UV dose in outdoor experiments.

Enhanced human tumor cell transplantability in a new congenic immunodeficient mouse; KSN-BNX

We introduced two mutant genes (beige; bg that induces the deficiency of natural killer (NK) activity andxid that decreases the production of immunoglobulin) into KSN nude mice with high reproductive performances. We produced KSNbg/bg(nu/nu) (KSN-bg), KSN-xid/xid(nu/nn) (KSN-xid), KSNxid/xid;bg/bg(nu/nu) (KSN-BNX) and KSN-nu/+ (KS) mice by backcross (cross-intercross method). All strains showed as high a reproductivity rate as the parental KSN mice. KSN-xid and KSN-BNX mice had a reduced percentage of B220 positive cells in the spleens compared to KSN and KSN-bg mice, but they showed increased percentages, of Thy-1 and asialo GM1 positive cells. The serum immunoglobulin concentrations of KSN-BNX were as low as KSN-xid. Both KSN-bg and KSN-BNX mice showed deficient NK activity in spleens, whereas KSN-xid mice showed an elevated NK activity. Compared to nude mice, the growth of both human tumor cell TCO-1 and BxPc-3 transplanted subcutaneously was enhanced in KSN-BNX mice. However Panc-1 cells that was rejected in nude mice was not accepted in KSN-BNX mice. Liver metastasis of human pancreatic tumor cells; Capan-1, BxPc-3 and MIAPaCa-2 were studied. No significant difference was observed in the percentage of metastasis formed mice between nude and KSN-BNX mice.