Details of a Researcher - Yoshikawa, Harunori

Papers - Yoshikawa, Harunori

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Linear ubiquitination triggers Amph-mediated T-tubule biogenesis

Kohei Kawaguchi, Yutaro Hama, Harunori Yoshikawa, Kohei Nishino, Kazuki Morimoto, Tsuyoshi Nakamura, Michiko Koizumi, Yuriko Sakamaki, Kota Abe, Soichiro Kakuta, Koichiro Ichimura, Fumiyo Ikeda, Hidetaka Kosako, Naonobu Fujita

2025.04

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Cover Feature: Sulfoxide‐Mediated Cys‐Trp‐Selective Bioconjugation that Enables Protein Labeling and Peptide Heterodimerization (ChemistryEurope 3‐4/2024)

Daishiro Kobayashi, Masaya Denda, Junya Hayashi, Kota Hidaka, Yutaka Kohmura, Takaaki Tsunematsu, Kohei Nishino, Harunori Yoshikawa, Kento Ohkawachi, Kiyomi Nigorikawa, Tetsuro Yoshimaru, Naozumi Ishimaru, Wataru Nomura, Toyomasa Katagiri, Hidetaka Kosako, Akira Otaka

ChemistryEurope 2024.05

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Proteomic and functional comparison between human induced and embryonic stem cells

Alejandro J. Brenes, Eva Griesser, Linda V. Sinclair, Lindsay Davidson, Alan R. Prescott, Francois Singh, Elizabeth K.J. Hogg, Carmen Espejo-Serrano, Hao Jiang, Harunori Yoshikawa, Melpomeni Platani, Jason Swedlow, Greg M. Findlay, Doreen A. Cantrell, Angus I. Lamond

eLife (eLife Sciences Publications, Ltd) 2024.02

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling, thereby avoiding ethical issues arising from the use of embryo-derived cells. However, despite clear similarities between the two cell types, it is likely they are not identical. In this study we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors. We find that while hESCs and hiPSCs express a near identical set of proteins, they show consistent quantitative differences in the expression levels of a wide subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs. The proteomic data show hiPSCs have significantly increased abundance of vital cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins, which correlated with phenotypic differences between hiPSCs and hESCs. Thus, higher levels of glutamine transporters correlated with increased glutamine uptake, while higher levels of proteins involved in lipid synthesis correlated with increased lipid droplet formation. Some of the biggest metabolic changes were seen in proteins involved in mitochondrial metabolism, with corresponding enhanced mitochondrial potential, shown experimentally using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins including ECM components and growth factors, some with known tumorigenic properties as well as proteins involved in the inhibition of the immune system. Our data indicate that reprogramming of human fibroblasts to iPSCs effectively restores protein expression in cell nuclei to a similar state to hESCs, but does not similarly restore the profile of cytoplasmic and mitochondrial proteins, with consequences for cell phenotypes affecting growth and metabolism. The data improve understanding of the molecular differences between induced and embryonic stem cells with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

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がん特殊化リボソームの同定と機能解析(Identification and characterization of cancer-specialized ribosomes)

常松貴明, 吉川治孝, 永尾瑠佳, 松澤鎮史, 大塚邦紘, 牛尾綾, 石丸直澄

日本病理学会会誌 ((一社)日本病理学会) 113 ( 1 ) 295 - 295 2024.02

ISSN 0300-9181

An allele-selective inter-chromosomal protein bridge supports monogenic antigen expression in the African trypanosome

Joana R. C. Faria, Michele Tinti, Catarina A. Marques, Martin Zoltner, Harunori Yoshikawa, Mark C. Field, David Horn

Nature Communications 2023.12

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Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics

Kohei Nishino, Harunori Yoshikawa, Kou Motani, Hidetaka Kosako

Journal of Proteome Research (American Chemical Society (ACS)) 2022.08

ISSN 1535-3893

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Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.

Takumi Maruhashi, Daisuke Sugiura, Il-Mi Okazaki, Kenji Shimizu, Takeo K Maeda, Jun Ikubo, Harunori Yoshikawa, Katsumi Maenaka, Naozumi Ishimaru, Hidetaka Kosako, Tatsuya Takemoto, Taku Okazaki

Immunity 55 ( 5 ) 912 - 924 2022.05

Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.

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Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE.

Harunori Yoshikawa, Kohei Nishino, Hidetaka Kosako

Journal of proteomics 258 104543 - 104543 2022.04

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE of in vitro kinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sites in vitro as those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases. SIGNIFICANCE: Label-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERK in vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells and in vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.

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Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states.

Francisco Rodriguez-Algarra, Robert A E Seaborne, Amy F Danson, Selin Yildizoglu, Harunori Yoshikawa, Pui Pik Law, Zakaryya Ahmad, Victoria A Maudsley, Ama Brew, Nadine Holmes, Mateus Ochôa, Alan Hodgkinson, Sarah J Marzi, Madapura M Pradeepa, Matthew Loose, Michelle L Holland, Vardhman K Rakyan

Genome biology 23 ( 1 ) 54 - 54 2022.02

BACKGROUND: Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed. RESULTS: Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%. CONCLUSIONS: Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

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Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC.

Harunori Yoshikawa, Ramasubramanian Sundaramoorthy, Daniel Mariyappa, Hao Jiang, Angus I Lamond

Bio-protocol 11 ( 15 ) e4106 2021.08

Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long ultracentrifugation to collecting and analyzing the fractions. Each of these steps in the SDG workflow can introduce potential technical variation that affects the reproducibility of gradient profiles between samples. To address these limitations, we have developed a flexible, alternative approach for analyzing polysomes and ribosomal subunits based on size-exclusion chromatography (SEC), termed 'Ribo Mega-SEC.' In comparison with the SDG method, Ribo Mega-SEC involves a single step using ultra-high-performance liquid chromatography (uHPLC). The entire workflow, from injecting the lysate to collecting the fractions, can be performed in as little as 15 min, with high reproducibility. By varying the pore size of the SEC column, polysomes and ribosomal subunits can be separated using extracts from either human or mouse cultured cell lines or from tissue samples, Drosophila embryos, or budding yeast. The resulting separated fractions are suitable for analysis using a wide range of subsequent analytical techniques including mass spectrometry (MS)-based proteomics, RNA-Seq, electron microscopy (EM), and multiple biochemical assays.

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Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation

Galloway, A., Kaskar, A., Ditsova, D., Atrih, A., Yoshikawa, H., Gomez-Moreira, C., Suska, O., Warminski, M., Grzela, R., Lamond, A.I., Darzynkiewicz, E., Jemielity, J., Cowling, V.H.

Nucleic Acids Research (Oxford University Press ({OUP})) 49 ( 12 ) 2021.06

ISSN 1362-4962

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Erosion of human X chromosome inactivation causes major remodeling of the iPSC proteome.

Alejandro J Brenes, Harunori Yoshikawa, Dalila Bensaddek, Bogdan Mirauta, Daniel Seaton, Jens L Hukelmann, Hao Jiang, Oliver Stegle, Angus I Lamond

Cell reports 35 ( 4 ) 109032 - 109032 2021.04

X chromosome inactivation (XCI) is a dosage compensation mechanism in female mammals whereby transcription from one X chromosome is repressed. Analysis of human induced pluripotent stem cells (iPSCs) derived from female donors identified that low levels of XIST RNA correlated strongly with erosion of XCI. Proteomic analysis, RNA sequencing (RNA-seq), and polysome profiling showed that XCI erosion resulted in amplified RNA and protein expression from X-linked genes, providing a proteomic characterization of skewed dosage compensation. Increased protein expression was also detected from autosomal genes without an mRNA increase, thus altering the protein-RNA correlation between the X chromosome and autosomes. XCI-eroded lines display an ∼13% increase in total cell protein content, with increased ribosomal proteins, ribosome biogenesis and translation factors, and polysome levels. We conclude that XCI erosion in iPSCs causes a remodeling of the proteome, affecting the expression of a much wider range of proteins and disease-linked loci than previously realized.

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LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA

Keiichi Izumikawa, Hideaki Ishikawa, Harunori Yoshikawa, Sally Fujiyama, Akira Watanabe, Hiroyuki Aburatani, Hiroyuki Tachikawa, Toshiya Hayano, Yutaka Miura, Toshiaki Isobe, Richard J Simpson, Li Li, Jinrong Min, Nobuhiro Takahashi

Nucleic Acids Research (Oxford University Press ({OUP})) 47 ( 19 ) 10357 - 10372 2019.09

Accepted

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

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Efficient analysis of mammalian polysomes in cells and tissues using ribo mega-SEC

Yoshikawa, H., Larance, M., Harney, D.J., Sundaramoorthy, R., Ly, T., Owen-Hughes, T., Lamond, A.I.

eLife 7 2018.08

Accepted, ISSN 2050-084X

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An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis

Liakath-Ali, Kifayathullah, Mills, Eric W., Sequeira, Ines, Lichtenberger, Beate M., Pisco, Angela Oliveira, Sipila, Kalle H., Mishra, Ajay, Yoshikawa, Harunori, Wu, Colin Chih-Chien, Ly, Tony, Lamond, Angus I., Adham, Ibrahim M., Green, Rachel, Watt, Fiona M.

Nature 556 ( 7701 ) 376 - 380 2018.04

Accepted, ISSN 0028-0836

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TDP-43 stabilises the processing intermediates of mitochondrial transcripts

Keiichi Izumikawa, Yuko Nobe, Harunori Yoshikawa, Hideaki Ishikawa, Yutaka Miura, Hiroshi Nakayama, Takashi Nonaka, Masato Hasegawa, Naohiro Egawa, Haruhisa Inoue, Kouki Nishikawa, Koji Yamano, Richard J. Simpson, Masato Taoka, Yoshio Yamauchi, Toshiaki Isobe, Nobuhiro Takahashi

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 7 ( 1 ) 2017.08

Accepted, ISSN 2045-2322

The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43-related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

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Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells

Hideaki Ishikawa, Harunori Yoshikawa, Keiichi Izumikawa, Yutaka Miura, Masato Taoka, Yuko Nobe, Yoshio Yamauchi, Hiroshi Nakayama, Richard J. Simpson, Toshiaki Isobe, Nobuhiro Takahashi

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 45 ( 6 ) 3437 - 3447 2017.04

Accepted, ISSN 0305-1048

Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E prerRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle. Knockdown of PARN or exogenous expression of an enzyme-dead PARN mutant (D28A) accumulated 18S-E in both the cytoplasm and nucleus. Moreover, expression of D28A accumulated 18S-E in Bystin-associated pre-40S particles, suggesting that the enzymatic activity of PARN is necessary for the release of 18S-E from Bystin-associated pre-40S particles. Finally, RNase H-based fragmentation analysis and 3'-sequence analysis of 18S-E species present in cells expressing wild-type PARN or D28A suggested that PARN degrades the extended regions encompassing nucleotides 5-44 at the 3' end of mature 18S rRNA. Our results reveal a novel role for PARN in ribosome biogenesis in human cells.

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Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA

Keiichi Izumikawa, Harunori Yoshikawa, Hideaki Ishikawa, Yuko Nobe, Yoshio Yamauchi, Sjaak Philipsen, Richard J. Simpson, Toshiaki Isobe, Nobuhiro Takahashi

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 44 ( 20 ) 9847 - 9859 2016.11

Accepted, ISSN 0305-1048

Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5' end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention.

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Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5 '-3 ' exoribonuclease) in the nucleoplasm

Shigeko Sato, Hideaki Ishikawa, Harunori Yoshikawa, Keiichi Izumikawa, Richard J. Simpson, Nobuhiro Takahashi

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 43 ( 21 ) 10397 - 10410 2015.12

Accepted, ISSN 0305-1048

Collaborator of alternative reading frame protein (CARF) associates directly with ARF, p53, and/or human double minute 2 protein (HDM2), a ubiquitin-protein ligase, without cofactors and regulates cell proliferation by forming a negative feedback loop. Although ARF, p53, and HDM2 also participate in the regulation of ribosome biogenesis, the involvement of CARF in this process remains unexplored. In this study, we demonstrate that CARF associates with 5'-3' exoribonuclease 2 (XRN2), which plays a major role in both the maturation of rRNA and the degradation of a variety of discarded pre-rRNA species. We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5' extended from of 45S/47S pre-rRNA and 5'-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus. This was also observed upon XRN2 knockdown. Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis. These observations suggest that CARF regulates early steps of pre-rRNA processing during ribosome biogenesis by controlling spatial distribution of XRN2 between the nucleoplasm and nucleolus.

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Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2

Masashi Saruhashi, Totan Kumar Ghosh, Kenta Arai, Yumiko Ishizaki, Kazuya Hagiwara, Kenji Komatsu, Yuh Shiwa, Keiichi Izumikawa, Harunori Yoshikawa, Taishi Umezawa, Yoichi Sakata, Daisuke Takezawa

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (NATL ACAD SCIENCES) 112 ( 46 ) E6388 - E6396 2015.11

Accepted, ISSN 0027-8424

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.

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Human nucleolar protein Nop52 (RRP1/NNP-1) is involved in site 2 cleavage in internal transcribed spacer 1 of pre-rRNAs at early stages of ribosome biogenesis

Harunori Yoshikawa, Hideaki Ishikawa, Keiichi Izumikawa, Yutaka Miura, Toshiya Hayano, Toshiaki Isobe, Richard J. Simpson, Nobuhiro Takahashi

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 43 ( 11 ) 5524 - 5536 2015.06

Accepted, ISSN 0305-1048

During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S.

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Comprehensive Analysis of RNA–Protein Complex Using Mass Spectrometry-Based Technology

Keiichi IZUMIKAWA, Hideaki ISHIKAWA, Harunori YOSHIKAWA, Toshiaki ISOBE, Nobuhiro TAKAHASHI

KAGAKU TO SEIBUTSU (Japan Society for Bioscience, Biotechnology, and Agrochemistry) 53 ( 12 ) 850 - 859 2015

ISSN 0453-073X

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Human cell growth regulator Ly-1 antibody reactive homologue accelerates processing of preribosomal RNA

Naoki Miyazawa, Harunori Yoshikawa, Satomi Magae, Hideaki Ishikawa, Keiichi Izumikawa, Goro Terukina, Ai Suzuki, Sally Nakamura-Fujiyama, Yutaka Miura, Toshiya Hayano, Wataru Komatsu, Toshiaki Isobe, Nobuhiro Takahashi

GENES TO CELLS (WILEY-BLACKWELL) 19 ( 4 ) 273 - 286 2014.04

Accepted, ISSN 1356-9597

Ribosome biogenesis is an essential process for cell growth and proliferation and is enhanced in cancer and embryonic stem cells. Mouse Ly-1 antibody reactive clone product (Lyar) is expressed at very high levels in many tumor, leukemia or embryonic stem cells; is a novel nucleolar protein with zinc-finger DNA-binding motifs and is involved in cell growth regulation. However, cellular function of Lyar remains unexplored. Here, we show that human homologue of Lyar (LYAR) accelerates ribosome biogenesis at the level of processing of preribosomal RNA (pre-rRNA). We show that LYAR is excluded from the nucleolus after actinomycin D treatment and is present in preribosomal fraction of the nuclear extract as well as in the fractions with 40S, 60S and 90S sedimentation coefficients. LYAR is required for processing of 47S/45S, 32S, 30S and 21S pre-rRNAs. In addition, we show that over-expression of LYAR increases cell proliferation without affecting the expression of c-Myc or p53. Combined, these results suggest that some rapidly growing cells enhance ribosome biogenesis by increasing the expression of LYAR.

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Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis

Hideaki Ishikawa, Yuko Nobe, Keiichi Izumikawa, Harunori Yoshikawa, Naoki Miyazawa, Goro Terukina, Natsuki Kurokawa, Masato Taoka, Yoshio Yamauchi, Hiroshi Nakayama, Toshiaki Isobe, Nobuhiro Takahashi

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 42 ( 4 ) 2708 - 2724 2014.02

Accepted, ISSN 0305-1048

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.

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Friend of Prmt1, FOP is a novel component of the nuclear SMN complex isolated using biotin affinity purification

Keiichi Izumikawa, Hideaki Ishikawa, Harunori Yoshikawa, Goro Terukina, Naoki Miyazawa, Hiroshi Nakayama, Yuko Nobe, Masato Taoka, Yoshio Yamauchi, Sjaak Philipsen, Toshiaki Isobe, Nobuhiro Takahashi

Journal of Proteomics and Bioinformatics (OMICS Publishing Group) 7 ( 4 ) 2014

Accepted, ISSN 0974-276X

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 complexes- 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 U5and U6 snRNAs in the nucleus, but not with Sm proteins, Gemin5, coilin, and U1 and U2snRNAs. 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, histone proteins, and various RNA-binding proteins. It is reported that FOP localizes mainly in the nuclear speckles, binds chromatin, and plays a role in mRNA transcriptional regulation. Our present data suggest that the nuclear SMN complex containing FOP participates in the process of mRNA post-transcriptional regulation. © 2013 Izumikawa K, et al.

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Human cell growth regulator LYAR is highly expressed in many tumors and accelerates ribosome biogenesis

Yoshikawa Harunori, Miyazawa Naoki, Magae Satomi, Ishikawa Hideaki, Izumikawa Keiichi, Terukina Goro, Miura Yutaka, Hayano Toshiya, Isobe Toshiaki, Watanabe Ryo, Aburatani Hiroyuki, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2014 78 - 78 2014

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Functional analysis of a novel component of the nuclear complex associated with survival motor neuron protein

Izumikawa Keiichi, Ishikawa Hideaki, Yoshikawa Harunori, Terukina Goro, Nobe Yuko, Nakayama Hiroshi, Taoka Masato, Yamauchi Yoshio, Isobe Toshiaki, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2014 76 - 76 2014

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Ribonucleoproteomics based approach identified U1-tfs in SMN-associated RNP complex and discovered a novel U1 snRNA metabolic pathway in human cell

Ishikawa Hideaki, Izumikawa Keiichi, Yoshikawa Harunori, Nobe Yuko, Yamauchi Yoshio, Taoka Masato, Nakayama Hiroshi, Isobe Toshiaki, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2014 79 - 79 2014

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Ribonucleoproteomic analysis on RNA modification and turnover

H Ishikawa, K Izumikawa, H Yoshikawa, Y Nobe, Y Yamauchi, M Taoka, H Nakayama, T Isobe, N Takahashi

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2014 36 - 36 2014

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[The regulatory mechanism underlying ribosomal subunit synthesis].

Yoshikawa, H., Izumikawa, K., Ishikawa, H., Takahashi, N.

Seikagaku. The Journal of Japanese Biochemical Society 85 ( 10 ) 861—870 2013

Accepted

Identification of a novel SMN1-associated RNP complex containing short form of U1 snRNA

Ishikawa Hideaki, Yoshikawa Harunori, Izumikawa Keiichi, Miura Yutaka, Taoka Masato, Nobe Yuko, Yamauchi Yoshio, Nakayama Hiroshi, Isobe Toshiaki, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2012 128 - 128 2012

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Molecular mechanism by which RNA helicase II/Guα regulates ribosome biogenesis

Yasumura Taiju, Yoshikawa Harunori, Ooshima Hitoshi, Benigno Valdez, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2012 129 - 129 2012

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Human cell growth regulator LYAR is highly expressed in many tumors and accelerates ribosome biogenesis.

Miyazawa Naoki, Yoshikawa Harunori, Magae Emi, Ishikawa Hideaki, Izumikawa Keiichi, Terukina Goro, Suzuki Ai, Fujiyama-Nakamura Sally, Miura Yutaka, Hayano Toshiya, Isobe Toshiaki, Watanabe Akira, Aburatani Hiroyuki, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2012 171 - 171 2012

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The role of nucleolar protein Nop52 in the pre-rRNA processing during human ribosome biogenesis revealed by proteomic approach

Yoshikawa Harunori, Kawasaki Miyuki, Hayano Toshiya, Izumikawa Keiichi, Ishikawa Hideaki, Miura Yutaka, Yamauchi Yoshio, Isobe Toshiaki, Takahashi Nobuhiro

Abstracts for Annual Meeting of Japanese Proteomics Society (Japanese Proteomics Society (Japan Human Proteome Organisation)) 2012 181 - 181 2012

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Splicing Factor 2-Associated Protein p32 Participates in Ribosome Biogenesis by Regulating the Binding of Nop52 and Fibrillarin to Preribosome Particles

Harunori Yoshikawa, Wataru Komatsu, Toshiya Hayano, Yutaka Miura, Keiichi Homma, Keiichi Izumikawa, Hideaki Ishikawa, Naoki Miyazawa, Hiroyuki Tachikawa, Yoshio Yamauchi, Toshiaki Isobe, Nobuhiro Takahashi

MOLECULAR & CELLULAR PROTEOMICS (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC) 10 ( 8 ) M110.006148 2011.08

Accepted, ISSN 1535-9476

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S preribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.006148, 1-23, 2011.

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Parvulin (Par14), a Peptidyl-Prolyl cis-trans Isomerase, Is a Novel rRNA Processing Factor That Evolved in the Metazoan Lineage

Sally Fujiyama-Nakamura, Harunori Yoshikawa, Keiichi Homma, Toshiya Hayano, Teruko Tsujimura-Takahashi, Keiichi Izumikawa, Hideaki Ishikawa, Naoki Miyazawa, Mitsuaki Yanagida, Yutaka Miura, Takashi Shinkawa, Yoshio Yamauchi, Toshiaki Isobe, Nobuhiro Takahashi

MOLECULAR & CELLULAR PROTEOMICS (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC) 8 ( 7 ) 1552 - 1565 2009.07

Accepted, ISSN 1535-9476

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage. Molecular & Cellular Proteomics 8: 1552-1565, 2009.

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