Hiroshi Nakayama

Japan Science and Technology Agency (JST), Edo, Tōkyō, Japan

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Publications (53)333.04 Total impact

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    ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene networks and participate in many physiological and pathological pathways. To date, miRNAs have been characterized mostly by genetic technologies, which have the advantages of being very sensitive and using high-throughput instrumentation; however, these techniques cannot identify most post-transcriptional modifications of miRNAs that would affect their functions. Herein, we report an analytical system for the direct identification of miRNAs that incorporates nano-flow liquid chromatography-high-resolution tandem mass spectrometry and RNA-sequence database searching. By introducing a spray-assisting device that stabilizes negative nano-electrospray ionization of RNAs and by searching an miRNA sequence database using the obtained tandem mass spectrometry data for RNA mixture, we successfully identified femtomole quantities of human cellular miRNAs and their 3'-terminal variants. This is the first report of a fully automated, and thus objective, tandem mass spectrometry-based analytical system that can be used to identify miRNAs.
    Analytical Chemistry 02/2015; DOI:10.1021/ac504378s · 5.83 Impact Factor
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    ABSTRACT: The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3' half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly.
    PLoS ONE 11/2014; 9(11):e112156. DOI:10.1371/journal.pone.0112156 · 3.53 Impact Factor
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    ABSTRACT: This study used global protein expression profiling to search for biomarkers to predict early recurrent hepatocellular carcinoma (HCC). HCC tissues surgically resected from patients with or without recurrence within 2 years (early recurrent) after surgery were compared with adjacent non-tumor tissue and with normal liver tissue. We used the PROTOMAP strategy for comparative profiling, which integrates denaturing polyacrylamide gel electrophoresis migratory rates and high-resolution, semi-quantitative mass spectrometry-based identification of in gel-digested tryptic peptides. PROTOMAP allows examination of global changes in the size, topography, and abundance of proteins in complex tissue samples. This approach identified 8,438 unique proteins from 45,708 non-redundant peptides and generated a proteome-wide map of changes in expression and proteolytic events potentially induced by intrinsic apoptotic/necrotic pathways. In the early-recurrent HCC tissue, 87 proteins were differentially expressed (≥20-fold) relative to the other tissues, 46 of which were up-regulated and/or specifically proteolyzed and 41 of which were down-regulated. This dataset consisted of proteins that fell into various functional categories, including signal transduction and cell organization and, notably, the major catalytic pathways responsible for liver function, such as the urea cycle and detoxification metabolism. We found that aberrant proteolysis appeared to occur frequently during recurrence of HCC in several key signal transducers, including STAT1 and -catenin. Further investigation of these proteins will facilitate the development of novel clinical applications.
    Journal of Proteome Research 06/2014; 13(11). DOI:10.1021/pr500262p · 5.00 Impact Factor
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    ABSTRACT: SMN (survival motor neuron protein) complexes are essential for the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). During the biogenesis, the SMN complexes bound to UsnRNPs are transported from the cytoplasm to the nucleus, and moved to Cajal body (bodies)/Gems (Cajal/Gems) where the SMN complex-UsnRNPs are subjected to additional chemical modifications and dissociated to the SMN complexes and the mature UsnRNPs. Although the mature UsnRNPs are assembled into spliceosome with newly transcribed pre-mRNA in the perichromatin fibrils at the chromatin, the role of the dissociated nuclear SMN complexes remains undetermined. In this study, we identified Friend of Prmt1 (FOP; chromatin target of Prmt1, CHTOP; C1orf77) as a novel component of the nuclear SMN complexes by the biotin affinity purification, coupled with the mass spectrometry-based protein identification. FOP was associated with SMN, Gemines 2, 3, 4, 6, and 8, unrip, and fragile X mental retardation 1 protein (FMR1), as well as with U5 and U6 snRNAs in the nucleus, but not with Sm proteins, Gemin5, coilin, and U1 and U2 snRNAs. Using the quantitative proteomic method with SILAC coupled with RNA interference, we also showed that FOP is required for the association of the SMN complexes with hnRNPs and histone proteins. FOP localizes mainly in the nuclear speckles, binds chromatin, and has roles in mRNA transcriptional regulation. Our present data suggest that the nuclear SMN complex containing FOP participates in the process of mRNA post-transcriptional regulation.
    Journal of Proteomics & Bioinformatics 12/2013; DOI:10.4172/jpb.S7-002
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    ABSTRACT: The U1 small nuclear ribonucleoprotein (snRNP) plays pivotal roles in pre-mRNA splicing and in regulating mRNA length and isoform expression; however, the mechanism of U1 snRNA quality control remains undetermined. Here, we describe a novel surveillance pathway for U1 snRNP biogenesis. Mass spectrometry-based RNA analysis showed that a small population of SMN complexes contains truncated forms of U1 snRNA (U1-tfs) lacking the Sm-binding site and stem loop 4 but containing a 7-monomethylguanosine 5' cap and a methylated first adenosine base. U1-tfs form a unique SMN complex, are shunted to processing bodies and have a turnover rate faster than that of mature U1 snRNA. U1-tfs are formed partly from the transcripts of U1 genes and partly from those lacking the 3' box elements or having defective SL4 coding regions. We propose that U1 snRNP biogenesis is under strict quality control: U1 transcripts are surveyed at the 3'-terminal region and U1-tfs are diverted from the normal U1 snRNP biogenesis pathway.
    Nucleic Acids Research 12/2013; DOI:10.1093/nar/gkt1271 · 8.81 Impact Factor
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    ABSTRACT: The ability of denaturing ion-paired reversed phase LC to separate RNA was assessed using macro-porous polystyrene-divinylbenzene resins as the stationary phase. Using the three stationary phases with different pore size and a mobile phase containing phosphate, we separated RNAs of 20-8000 nucleotides with extremely high sensitivity, e.g., 50pg for an RNA 20 nucleotides in length, S/N=5. The method was used to separate non-coding RNAs obtained from biological sources and is suited for use with direct MS-based chemical characterization.
    Journal of Chromatography A 09/2013; DOI:10.1016/j.chroma.2013.09.021 · 4.26 Impact Factor
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    ABSTRACT: Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect-bacterium symbiotic association. The bean bug Riptortus pedestris harbors a beneficial and specific gut symbiont of the β-proteobacterial genus Burkholderia, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured Burkholderia detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic Burkholderia. Among major PHA synthesis genes, phaB and phaC were disrupted by homologous recombination together with the phaP gene, whereby ΔphaB, ΔphaC, and ΔphaP mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in ΔphaB and ΔphaC, but only moderately in ΔphaP. In symbiosis, the host insects infected with ΔphaB and ΔphaC exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with ΔphaB and ΔphaC were restored by complementation of the mutants with plasmids encoding a functional phaB/phaC gene. Retention analysis of the plasmids revealed positive selection acting on the functional phaB/phaC in symbiosis. These results indicate that the PHA synthesis genes of the Burkholderia symbiont are required for normal symbiotic association with the Riptortus host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.
    Proceedings of the National Academy of Sciences 06/2013; 110(26). DOI:10.1073/pnas.1303228110 · 9.81 Impact Factor
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    ABSTRACT: Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN-) as its sole energy source. Previously, we showed that SCN- is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN- assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the β-class carbonic anhydrases (β-CAs). Although the crystal structure including the catalytic site resembles those of the β-CAs, CO2 hydration activity of COSase is negligible compared to those of the β-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The kcat/Km value, 9.6 × 105 s-1 M-1, is comparable to those of the β-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of β-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS.
    Journal of the American Chemical Society 02/2013; 135(10). DOI:10.1021/ja307735e · 11.44 Impact Factor
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    Hiroshi Nakayama, Kenji Kurokawa, Bok Luel Lee
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    ABSTRACT: Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor onto bacterial cell membranes. These proteins play important roles in a wide variety of bacterial physiological processes including virulence, and induce innate immune reactions by functioning as ligands of the mammalian Toll-like receptor 2. Here, we review recent advances in our understanding of bacterial lipoprotein structure, biosynthesis, and structure-function relationships between bacterial lipoproteins and Toll-like receptor 2. Notably, 40 years after the first report of the triacyl structure of Braun's lipoprotein in Escherichia coli, recent intensive mass spectrometry-based analyses have led to the discovery of three new lipidated structures of lipoproteins in monoderm bacteria: the lyso, N-acetyl, and peptidyl forms. Moreover, bacterial lipoprotein structure is believed to be constant in each bacterium; however, lipoprotein structures in Staphylococcus aureus vary between the diacyl and triacyl forms depending on environmental conditions. Thus, the lipidation state of bacterial lipoproteins, particularly in monoderm bacteria, is more complex than previously thought. © 2012 The Authors Journal compilation © 2012 FEBS.
    FEBS Journal 10/2012; DOI:10.1111/febs.12041 · 3.99 Impact Factor
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    ABSTRACT: In eukaryotic cells, components of messenger ribonucleoproteins (mRNPs) are often detected in cytoplasmic granules, such as processing bodies (P-bodies) and stress granules (SGs) where translationally repressed mRNAs accumulate. RAP55A, which is an RNA binding component of mRNPs, acts as a translational repressor and localizes to P-bodies and SGs. We found here that a homologous protein RAP55B also localized to P-bodies when expressed in human cultured cells. When RAP55A or RAP55B was highly expressed in the cells, they induced the formation of SG-like large cytoplasmic mRNP granules that contained both P-body and SG components, indicating that RAP55 is important for the assembly of cytoplasmic mRNP granules. In addition, we found that RAP55A associated with protein arginine methyltransferases PRMT1 and PRMT5. Multiple arginine residues of RAP55A were indeed asymmetrically dimethylated in the cell and PRMT1 was shown to be a component of large mRNP granules induced by RAP55A overexpression. Although PRMT1 did not accumulate in P-bodies, siRNA-mediated knockdown of PRMT1 impaired the localization of RAP55A to P-bodies, while other components were still retained in these structures. Thus, our data indicate that RAP55 is important for the assembly of cytoplasmic mRNP granules and that PRMT1 is required for RAP55A to localize to P-bodies.
    RNA biology 05/2012; 9(5):610-23. DOI:10.4161/rna.19527 · 5.38 Impact Factor
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    ABSTRACT: Bacterial lipoproteins are believed to exist in only one specific lipid-modified structure, such as the diacyl form or the triacyl form, in each bacterium. In the case of Staphylococcus aureus, recent extensive matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis revealed that S. aureus lipoproteins exist in the α-aminoacylated triacyl form. Here, we discovered conditions that induce the accumulation of diacyl lipoproteins that lack α-aminoacylation in S. aureus. The accumulation of diacyl lipoproteins required a combination of conditions, including acidic pH and a post-logarithmic-growth phase. High temperatures and high salt concentrations additively accelerated the accumulation of the diacyl lipoprotein form. Following a post-logarithmic-growth phase where S. aureus MW2 cells were grown at pH 6, SitC lipoprotein was found almost exclusively in its diacyl structure rather than in its triacyl structure. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.
    Journal of bacteriology 03/2012; 194(13):3299-306. DOI:10.1128/JB.00314-12 · 2.69 Impact Factor
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    ABSTRACT: Bacterial lipoproteins/lipopeptides inducing host innate immune responses are sensed by mammalian Toll-like receptor 2 (TLR2). These bacterial lipoproteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid. The presence of diacylated lipoproteins has been predicted in low-GC content gram-positive bacteria and mycoplasmas based on the absence of one modification enzyme in their genomes; however, we recently determined triacylated structures in low-GC gram-positive Staphylococcus aureus, raising questions about the actual lipoprotein structure in other low-GC content gram-positive bacteria. Here, through intensive MS analyses, we identified a novel and unique bacterial lipoprotein structure containing an N-acyl-S-monoacyl-glyceryl-cysteine (named the lyso structure) from low-GC gram-positive Enterococcus faecalis, Bacillus cereus, Streptococcus sanguinis, and Lactobacillus bulgaricus. Two of the purified native lyso-form lipoproteins induced proinflammatory cytokine production from mice macrophages in a TLR2-dependent and TLR1-independent manner but with a different dependence on TLR6. Additionally, two other new lipoprotein structures were identified. One is the "N-acetyl" lipoprotein structure containing N-acetyl-S-diacyl-glyceryl-cysteine, which was found in five gram-positive bacteria, including Bacillus subtilis. The N-acetyl lipoproteins induced the proinflammatory cytokines through the TLR2/6 heterodimer. The other was identified in a mycoplasma strain and is an unusual diacyl lipoprotein structure containing two amino acids before the lipid-modified cysteine residue. Taken together, our results suggest the existence of novel TLR2-stimulating lyso and N-acetyl forms of lipoproteins that are conserved in low-GC content gram-positive bacteria and provide clear evidence for the presence of yet to be identified key enzymes involved in the bacterial lipoprotein biosynthesis.
    Journal of Biological Chemistry 02/2012; 287(16):13170-81. DOI:10.1074/jbc.M111.292235 · 4.60 Impact Factor
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    ABSTRACT: To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.
    Journal of Biological Chemistry 12/2011; 287(5):3138-46. DOI:10.1074/jbc.M111.277921 · 4.60 Impact Factor
  • Hiroshi Nakayama, Nobuhiro Takahashi, Toshiaki Isobe
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    ABSTRACT: Mass spectrometry (MS) allows the sensitive and direct characterization of biological macromolecules and therefore has the potential to complement the more conventional genetic and biochemical methods used for RNA characterization. Although MS has been used much less frequently for RNA research than it has been for protein research, recent technical improvements in both instrumentation and software make MS a powerful tool for RNA analysis because it can now be used to sequence, quantify, and chemically analyze RNAs. Mass spectrometry is particularly well suited for the characterization of RNAs associated with ribonucleoprotein complexes. This review focuses on the software and databases that can be used for MS-based RNA studies. Software for the processing of raw mass spectra, the identification and characterization of RNAs by mass mapping, de novo sequencing, and tandem MS-based database searching are available.
    Mass Spectrometry Reviews 11/2011; 30(6):1000-12. DOI:10.1002/mas.20325 · 8.05 Impact Factor
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    ABSTRACT: Bacterial lipoproteins are known to be diacylated or triacylated and activate mammalian immune cells via Toll-like receptor 2/6 or 2/1 heterodimer. Because the genomes of low G+C content gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apolipoprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determination of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host-microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipoprotein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resistant fatty acid. Further MS/MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to α-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form.
    FEBS Journal 03/2011; 278(5):716-28. DOI:10.1111/j.1742-4658.2010.07990.x · 3.99 Impact Factor
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    ABSTRACT: In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.
    Journal of Biological Chemistry 10/2010; 285(43):32937-45. DOI:10.1074/jbc.M110.144014 · 4.60 Impact Factor
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    ABSTRACT: Although current mass spectrometry-based proteomics technology allows for high-throughput analysis of protein components in functional ribonucleoprotein complexes, this technology has had limited application to studies of RNA itself. Here we present a protocol for RNA analysis using polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry. Specifically, RNAs of interest are subjected to polyacrylamide gel electrophoresis and stained with a fluorescent dye, and RNAs in gel bands are digested with nuclease and then analyzed directly liquid chromatography-mass spectrometry, resulting in highly accurate mass values and reliable information on post-transcriptional modifications. We demonstrate that the method can be applied to the identification and chemical analysis of small RNAs in mouse embryonic stem cell extracts and of small RNAs in the spliceosomal ribonucleoprotein complex pulled down from yeast cells using a tagged protein cofactor as bait. The protocol is relatively simple and allowed us to identify not only three novel methylated nucleotide residues of RNase P RNA, U6 snRNA, and 7SL RNA prepared from mouse ES cells but also various 3'-end forms of U4, U5S, and U6 snRNAs isolated from the yeast spliceosome at the femtomole level. The method is thus a convenient tool for direct analysis of RNAs in various cellular ribonucleoprotein complexes, particularly for the analysis of post-transcriptional modifications and metabolic processing of RNA.
    Analytical Chemistry 09/2010; 82(18):7795-803. DOI:10.1021/ac101623j · 5.83 Impact Factor
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    ABSTRACT: A variety of unique codons have been employed to expand the genetic code. The use of the opal (UGA) codon is promising, but insufficient information is available about the UGA suppression approach, which facilitates the incorporation of non-natural amino acids through suppression of the UGA codon. In this study, the UGA codon was used to incorporate 4-iodo-l-phenylalanine into position 32 of the Ras protein in an Escherichia coli cell-free translation system. The undesired incorporation of tryptophan in response to the UGA codon was completely repressed by the addition of indolmycin. The minor amount (3%) of contaminating 4-bromo-l-phenylalanine in the building block 4-iodo-l-phenylalanine led to the significant incorporation of 4-bromo-l-phenylalanine (21%), and this problem was solved by using a purified 4-iodo-l-phenylalanine sample. Optimization of the incubation time was also important, since the undesired incorporation of free phenylalanine increased during the cell-free translation reaction. The 4-iodo-l-phenylalanine residue can be used for the chemoselective modification of proteins. This method will contribute to advancements in protein engineering studies with non-natural amino acid substitutions.
    Journal of Biochemistry 08/2010; 148(2):179-87. DOI:10.1093/jb/mvq051 · 3.07 Impact Factor
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    ABSTRACT: Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.
    The EMBO Journal 11/2009; 28(24):3868-78. DOI:10.1038/emboj.2009.343 · 10.75 Impact Factor
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    ABSTRACT: We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a approximately 21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.
    Nucleic Acids Research 10/2009; 37(21):e140. DOI:10.1093/nar/gkp732 · 8.81 Impact Factor

Publication Stats

2k Citations
333.04 Total Impact Points

Institutions

  • 2010–2014
    • Japan Science and Technology Agency (JST)
      Edo, Tōkyō, Japan
  • 2002–2014
    • RIKEN
      • Antibiotics Laboratory
      Вако, Saitama, Japan
  • 2012
    • Pusan National University
      • College of Pharmacy
      Pusan, Busan, South Korea
  • 2011
    • Kanazawa University
      • Graduate School of Medical Sciences
      Kanazawa, Ishikawa, Japan
  • 2006
    • Tokyo University of Agriculture and Technology
      • United Graduate School of Agricultural Science
      Edo, Tōkyō, Japan
  • 2002–2004
    • Tokyo Metropolitan University
      • Department of Chemistry
      Tokyo, Tokyo-to, Japan
  • 2003
    • Utsunomiya University
      Totigi, Tochigi, Japan
  • 2001
    • National Institute of Advanced Industrial Science and Technology
      • Biomedicinal Information Research Center
      Tokyo, Tokyo-to, Japan