Kohei Nishino, Harunori Yoshikawa, Kou Motani and Hidetaka Kosako : Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics., Journal of Proteome Research, 2022.
(要約)
Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.
Takumi Maruhashi, Daisuke Sugiura, Il-mi Okazaki, Kenji Shimizu, K Takeo Maeda, Jun Ikubo, Harunori Yoshikawa, Katsumi Maenaka, Naozumi Ishimaru, Hidetaka Kosako, Tatsuya Takemoto and Taku Okazaki : Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity., Immunity, Vol.55, No.5, 912-924.e8, 2022.
(要約)
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 invitro and invivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced Tcell suppression invitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity invitro. 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.
(キーワード)
Animals / Antigens, CD / Autoimmunity / Histocompatibility Antigens Class II / Ligands / Mice / Mice, Inbred C57BL / Mice, Inbred NOD / Neoplasms / Peptides / T-Lymphocytes
Harunori Yoshikawa, Kohei Nishino and Hidetaka Kosako : Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE., Journal of Proteomics, Vol.258, 2022.
(要約)
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.
(キーワード)
Animals / Chromatography, Liquid / Electrophoresis, Polyacrylamide Gel / Extracellular Signal-Regulated MAP Kinases / Mice / NIH 3T3 Cells / Phosphoproteins / Phosphorylation / Pyridines / Tandem Mass Spectrometry
Francisco Rodriguez-Algarra, E Robert A Seaborne, F Amy Danson, Selin Yildizoglu, Harunori Yoshikawa, Pik Pui Law, Zakaryya Ahmad, A Victoria Maudsley, Ama Brew, Nadine Holmes, Mateus Ochôa, Alan Hodgkinson, J Sarah Marzi, M Madapura Pradeepa, Matthew Loose, L Michelle Holland and K Vardhman Rakyan : Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states., Genome Biology, Vol.23, No.1, 2022.
(要約)
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.
Harunori Yoshikawa, Ramasubramanian Sundaramoorthy, Daniel Mariyappa, Hao Jiang and I Angus Lamond : Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC., Bio-protocol, Vol.11, No.15, 2021.
Alison Galloway, Aneesa Kaskar, Dimitrinka Ditsova, Abdelmadjid Atrih, Harunori Yoshikawa, Carolina Gomez-Moreira, Olga Suska, Marcin Warminski, Renata Grzela, I Angus Lamond, Edward Darzynkiewicz, Jacek Jemielity and H Victoria Cowling : Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation., Nucleic Acids Research, 2021.
(要約)
The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.
J Alejandro Brenes, Harunori Yoshikawa, Dalila Bensaddek, Bogdan Mirauta, Daniel Seaton, L Jens Hukelmann, Hao Jiang, Oliver Stegle and I Angus Lamond : Erosion of human X chromosome inactivation causes major remodeling of the iPSC proteome., Cell Reports, Vol.35, No.4, 2021.
(要約)
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.
Keiichi Izumikawa, Hideaki Ishikawa, Harunori Yoshikawa, Sally Fujiyama, Akira Watanabe, Hiroyuki Aburatani, Hiroyuki Tachikawa, Toshiya Hayano, Yutaka Miura, Toshiaki Isobe, J Richard Simpson, Li Li, Jinrong Min and Nobuhiro Takahashi : LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA., Nucleic Acids Research, Vol.47, No.19, 10357-10372, 2019.
(要約)
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.
Harunori Yoshikawa, Mark Larance, J Dylan Harney, Ramasubramanian Sundaramoorthy, Tony Ly, Tom Owen-Hughes and I Angus Lamond : Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC., eLife, Vol.7, No.e36530, 2018.
(要約)
We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.
(キーワード)
Amino Acids / Animals / Cell Line / Chromatography, High Pressure Liquid / Humans / Mice / Polyribosomes / Protein Biosynthesis / Proteomics / Ribosomes
Kifayathullah Liakath-Ali, W Eric Mills, Inês Sequeira, M Beate Lichtenberger, Oliveira Angela Pisco, H Kalle Sipilä, Ajay Mishra, Harunori Yoshikawa, Chih-Chien Colin Wu, Tony Ly, I Angus Lamond, M Ibrahim Adham, Rachel Green and M Fiona Watt : An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis., Nature, Vol.556, No.7701, 376-380, 2018.
(要約)
One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes
Keiichi Izumikawa, Yuko Nobe, Harunori Yoshikawa, Hideaki Ishikawa, Yutaka Miura, Hiroshi Nakayama, Takashi Nonaka, Masato Hasegawa, Naohiro Egawa, Haruhisa Inoue, Kouki Nishikawa, Koji Yamano, J Richard Simpson, Masato Taoka, Yoshio Yamauchi, Toshiaki Isobe and Nobuhiro Takahashi : TDP-43 stabilises the processing intermediates of mitochondrial transcripts., Scientific Reports, Vol.7, No.1, 2017.
(要約)
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.
(キーワード)
Cell Line / DNA-Binding Proteins / Gene Expression / Genes, Mitochondrial / Humans / Mitochondria / Protein Binding / Protein Transport / RNA Processing, Post-Transcriptional / RNA Stability / RNA, Transfer / Transcription, Genetic
Hideaki Ishikawa, Harunori Yoshikawa, Keiichi Izumikawa, Yutaka Miura, Masato Taoka, Yuko Nobe, Yoshio Yamauchi, Hiroshi Nakayama, J Richard Simpson, Toshiaki Isobe and Nobuhiro Takahashi : Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells., Nucleic Acids Research, Vol.45, No.6, 3437-3447, 2017.
(要約)
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 pre-rRNA (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.
Keiichi Izumikawa, Harunori Yoshikawa, Hideaki Ishikawa, Yuko Nobe, Yoshio Yamauchi, Sjaak Philipsen, J Richard Simpson, Toshiaki Isobe and Nobuhiro Takahashi : Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA., Nucleic Acids Research, Vol.44, No.20, 9847-9859, 2016.
(要約)
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.
(キーワード)
5' Untranslated Regions / Alternative Splicing / Cell Line / Gene Expression / Gene Expression Regulation / Gene Order / Homeostasis / Humans / Introns / Models, Biological / Nonsense Mediated mRNA Decay / Nuclear Proteins / Protein Binding / Protein Interaction Domains and Motifs / RNA Splicing / RNA, Messenger / Transcription Factors
Shigeko Sato, Hideaki Ishikawa, Harunori Yoshikawa, Keiichi Izumikawa, Richard J. Simpson and Nobuhiro Takahashi : Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5 '-3 ' exoribonuclease) in the nucleoplasm, Nucleic Acids Research, Vol.43, No.21, 10397-10410, 2015.
(要約)
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.
Masashi Saruhashi, Totan Ghosh Kumar, Kenta Arai, Yumiko Ishizaki, Kazuya Hagiwara, Kenji Komatsu, Yuh Shiwa, Keiichi Izumikawa, Harunori Yoshikawa, Taishi Umezawa, Yoichi Sakata and Daisuke Takezawa : Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2., Proceedings of the National Academy of Sciences of the United States of America, Vol.112, No.46, E6388-96, 2015.
(要約)
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.
Harunori Yoshikawa, Hideaki Ishikawa, Keiichi Izumikawa, Yutaka Miura, Toshiya Hayano, Toshiaki Isobe, J Richard Simpson and Nobuhiro Takahashi : 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., Nucleic Acids Research, Vol.43, No.11, 5524-5536, 2015.
(要約)
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.
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 and Nobuhiro Takahashi : Human cell growth regulator Ly-1 antibody reactive homologue accelerates processing of preribosomal RNA., Genes to Cells, Vol.19, No.4, 273-286, 2014.
(要約)
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.
Hideaki Ishikawa, Yuko Nobe, Keiichi Izumikawa, Harunori Yoshikawa, Naoki Miyazawa, Goro Terukina, Natsuki Kurokawa, Masato Taoka, Yoshio Yamauchi, Hiroshi Nakayama, Toshiaki Isobe and Nobuhiro Takahashi : Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis., Nucleic Acids Research, Vol.42, No.4, 2708-2724, 2013.
(要約)
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.
(キーワード)
Adenosine / Cytoplasm / Guanosine / Mass Spectrometry / Methylation / RNA Stability / RNA, Small Nuclear / Ribonucleoproteins, Small Nuclear / SMN Complex Proteins
Harunori Yoshikawa, Wataru Komatsu, Toshiya Hayano, Yutaka Miura, Keiichi Homma, Keiichi Izumikawa, Hideaki Ishikawa, Naoki Miyazawa, Hiroyuki Tachikawa, Yoshio Yamauchi, Toshiaki Isobe and Nobuhiro Takahashi : Splicing factor 2-associated protein p32 participates in ribosome biogenesis by regulating the binding of Nop52 and fibrillarin to preribosome particles., Molecular & Cellular Proteomics, Vol.10, No.8, 2011.
(要約)
Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome 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.
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 and Nobuhiro Takahashi : Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage., Molecular & Cellular Proteomics, Vol.8, No.7, 1552-1565, 2009.
(要約)
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.
Harunori Yoshikawa : Efficient analysis of translation-active ribosomes in cells and tissues, The 16th International Symposium of the Institute Network for Biomedical Sciences & KEY FORUM 2021 International Symposium, online, Kumamoto, Japan, Nov. 2021.
2.
Harunori Yoshikawa and AI Lamond : Full Spectrum Complexome Analyses: Towards the comprehensive characterisation of cellular protein complexes by Size Exclusion Chromatography, British Society for Proteome Research Annual Scientific Meeting, Southampton, UK, Jul. 2019.
3.
Harunori Yoshikawa, K Kilgour and AI Lamond : Full spectrum complexome analyses: Towards the comprehensive characterisation of cellular protein complexes by SEC-uHPLC, EMBLWellcome Genome Campus Conference: Proteomics in Cell Biology and Disease Mechanisms, Heidelberg, Germany, Feb. 2019.
𠮷川 治孝, 小迫 英尊 : Protein Correlation Profilingによる細胞内巨大タンパク質複合体の解析, 日本プロテオーム学会2022年大会, 2022年8月.
7.
𠮷川 治孝 : Towards the comprehensive characterisation of cellular protein complexes by Size Exclusion Chromatography, 日本プロテオーム学会2019年大会・第70回日本電気泳動学会総会, 2019年7月.
8.
𠮷川 治孝 : p32, FBL and Nop52 are involved in splitting step of pre-90S into pre-40S and pre-60S particles during human ribosome biogenesis, Ribosome Meeting, 2012年3月.
Harunori Yoshikawa : Faster and more efficient approach for isolating ribosomes and nucleolar pre-ribosomal particles, RNA public seminar series, The Centre for Integrative Biology of Toulouse (CBI), France, online, Apr. 2022.
Harunori Yoshikawa : Faster, more efficient and convenient approach for translation and ribosome biogenesis study, Institute for Cell & Molecular BioSciences, Newcastle University, UK, Oct. 2019.