Biomedical subjects

Toshiya Hayano

Publications and source records attributed to Toshiya Hayano.

9 recordsLinked to original sources

PubMed2006

NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47.

Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.

Amino Acid Sequence↗

PubMed2006

Potential roles for ubiquitin and the proteasome during ribosome biogenesis.

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.

Cell Line↗

PubMed2005

ERp57 binds competitively to protein disulfide isomerase and calreticulin.

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS-PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K(D) value of PDI binding to ERp57 was calculated as 5.46x10(-6)M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.

Animals↗

PubMed2003

Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes.

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.

Chromosomal Proteins, Non-Histone↗

PubMed2003

Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome.

Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.

Amino Acid Motifs↗

PubMed2003

Proteomics: advanced technology for the analysis of cellular function.

Proteomics developed initially from the decade-long study of comprehensive protein visualization on two-dimensional electrophoresis gels has been expanded by mass spectrometry and the growth in searchable sequence databases. Currently, by use of more sophisticated technology such as a combination of multidimensional chromatography and mass spectrometry, thousands of proteins can automatically be identified in a day along with semiquantitative information on differential-protein expression. As with differential gene expression by cDNA-chips, the differential-protein analysis is useful for monitoring and identifying proteins involved in various physiological changes in cells or organisms, although the analysis alone does not necessarily provide information regarding the cause of the change or the function of the proteins. However, proteomics also provides the tools to expand into more sophisticated biochemical approaches, such as the study of protein interactions that can be determined directly by performing a pull-down assay with a bait protein followed by mass spectrometric identification of the bound proteins. Proteomics, thus, is useful for both large-scale surveys of proteins and detailed studies of the functional relationships among the proteins of interest. Certainly this approach can be applicable to the assessment of amino acid adequacy and safety.

Animals↗

PubMed2002

Phosphorylation-dependent interaction of kinesin light chain 2 and the 14-3-3 protein.

The protein 14-3-3 is a key regulator in a cell signaling pathway mediated by protein phosphorylation. To identify the cellular targets of this protein systematically, we have employed a proteomic approach: protein components pulled down from PC12 cells stably expressing a myc-tagged 14-3-3eta isoform were analyzed by means of SDS-PAGE and mass spectrometry. This procedure allowed us to identify more than 30 proteins that include various known and unknown targets of the 14-3-3 protein. Among them are several proteins in the membrane traffic pathway, such as the heavy and light chains (KHC/KIF5B and KLC2) of conventional kinesin, a heterotetrameric mechanochemical motor involved in the ATP-dependent movement of vesicles and organelles along microtubules. Subsequent analysis showed that 14-3-3 directly binds to kinesin heterodimers through interaction with KLC2 and that this interaction is dependent on the phosphorylation of KLC2. Studies on the interaction between 14-3-3 and KLC2 variants expressed in cultured cells coupled with mass spectrometric analysis proved that Ser575 is the site of phosphorylation in KLC2 that is responsible for the in vivo interaction with the 14-3-3 protein. These data add KLC2 to the growing list of 14-3-3 targets, and suggest a role of 14-3-3 in the phosphorylation-regulated cellular transport of vesicles and organelles.

14-3-3 Proteins↗

PubMed2002

Isolation and proteomic characterization of human Parvulin-associating preribosomal ribonucleoprotein complexes.

Human parvulin (hParvulin; Par14/EPVH) belongs to the third family of peptidylprolyl cis-trans isomerases that exhibit an enzymatic activity of interconverting the cis-trans conformation of the prolyl peptide bond, and shows sequence similarity to the regulator enzyme for cell cycle transitions, human Pin1. However, the cellular function of hParvulin is entirely unknown. Here, we demonstrate that hParvulin associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes, which contain preribosomal RNAs, at least 26 ribosomal proteins, and 26 trans-acting factors involved in rRNA processing and assembly at an early stage of ribosome biogenesis. Since an amino-terminal domain of hParvulin, which is proposed to be a putative DNA-binding domain, was alone sufficient to associate in principle with the pre-rRNP complexes, the association is probably through protein-RNA interaction. In addition, hParvulin co-precipitated at least 10 proteins not previously known to be involved in ribosome biogenesis. Coincidentally, most of these proteins are implicated in regulation of microtubule assembly or nucleolar reformation during the mitotic phase of the cell. Thus, these results, coupled with the preferential nuclear localization of hParvulin, suggest that hParvulin may be involved in ribosome biogenesis and/or nucleolar re-assembly of mammalian cells.

Animals↗

PubMedDate not supplied

Proteomic snapshot analyses of preribosomal ribonucleoprotein complexes formed at various stages of ribosome biogenesis in yeast and mammalian cells.

Proteomic technologies powered by advancements in mass spectrometry and bioinformatics and coupled with accumulated genome sequence data allow a comprehensive study of cell function through large-scale and systematic protein identifications of protein constituents of the cell and tissues, as well as of multi-protein complexes that carry out many cellular function in a higher-order network in the cell. One of the most extensively analyzed cellular functions by proteomics is the production of ribosome, the protein-synthesis machinery, in the nucle(ol)us--the main site of ribosome biogenesis. The use of tagged proteins as affinity bait, coupled with mass spectrometric identification, enabled us to isolate synthetic intermediates of ribosomes that might represent snapshots of nascent ribosomes at particular stages of ribosome biogenesis and to identify their constituents--some of which showed dynamic changes for association with the intermediates at various stages of ribosome biogenesis. In this review, in conjunction with the results from yeast cells, our proteomic approach to analyze ribosome biogenesis in mammalian cells is described.

Animals↗