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Genes to Cells: Devoted to Molecular & Cellular Mechanisms

Shohei Kitano, Hikaru Kurasawa, Yasunori Aizawa
Transposons are major drivers of mammalian genome evolution. To obtain new insights into the contribution of transposons to the regulation of protein translation, we here examined how transposons affected the genesis and function of upstream open reading frames (uORFs), which serve as cis-acting elements to regulate translation from annotated ORFs (anORFs) located downstream of the uORFs in eukaryotic mRNAs. Among 39,786 human uORFs, 3,992 had ATG trinucleotides of a transposon origin, termed "transposon-derived upstream ATGs" or TuATGs...
February 15, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Rie Teshima, Katsuhiro Hanada, Junko Akada, Kenji Kawano, Yoshio Yamaoka
Periodontal disease, an inflammatory disease, is caused by infection with periodontal pathogens. Long-term periodontal disease increases the risk of oral carcinogenesis. Similar to other peptic cancers, oral carcinogenesis also requires multiple genome instabilities; however, the risk factors related to the accumulation of genome instabilities are poorly understood. Here, we suggested that specific periodontal pathogens may increase the risk of genome instability. Accordingly, we screened several periodontal pathogens based on the ability to induce DNA double-strand breaks (DSBs) in host cells...
February 14, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Nanami Akai, Tatsushi Igaki, Shizue Ohsawa
Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild-type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, we established two assay systems for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3-RNAi-expressing cells) system in which RpS3-RNAi-expressing wing pouch cells are eliminated from wild-type wing disc and (ii) the SURE (supercompetition of RpS3-expressing clones in RpS3/+ tissue) system in which RpS3-over-expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells...
February 12, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Yuki Ueda, Shin Kedashiro, Masahiro Maruoka, Kiyohito Mizutani, Yoshimi Takai
The immunoglobulin (Ig)-like cell adhesion molecule nectin-like molecule (Necl)-5/poliovirus receptor is up-regulated in many types of cancer cells and implicated in their abnormally enhanced cell proliferation and movement. We previously showed that Necl-5 cis-interacts with the platelet-derived growth factor (PDGF) receptor β through the extracellular region and enhances its signaling. Although this cis-interaction does not affect the PDGF-induced tyrosine phosphorylation of the receptor, the interaction of the cytoplasmic region of Necl-5 with sprouty2 and the regulation of its activity are required for the enhancement of the PDGF receptor β signaling by Necl-5...
February 12, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Shotaro Sakakibara, Tomohiko Maruo, Muneaki Miyata, Kiyohito Mizutani, Yoshimi Takai
The apical junctional complex consists of adherens junctions (AJs) and tight junctions (TJs) in polarized epithelial cells, which are attached to each other to form a sheet. Actin filaments (F-actin) are associated with AJs and TJs and required for the formation and maintenance of this complex. l-Afadin is an F-actin-binding protein, which is localized at AJs through binding to the cell adhesion molecule nectin, and regulates the formation of AJs and TJs. However, the role of the F-actin-binding activity of l-afadin for the formation of the apical junctional complex remains unknown...
February 12, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Kohei Nukina, Akiyo Hayashi, Yasushi Shiomi, Kaoru Sugasawa, Motoaki Ohtsubo, Hideo Nishitani
CRL4Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C-terminal half region of Cdt2 (Cdt2-18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo...
February 9, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Tetsushi Sakuma, Keiji Mochida, Shota Nakade, Toru Ezure, Sachi Minagawa, Takashi Yamamoto
Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones...
February 9, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Miki Kinoshita, Keiichi Namba, Tohru Minamino
FliG is a rotor protein of the bacterial flagellar motor. FliG consists of FliGN , FliGM and FliGC domains. Intermolecular FliGM -FliGC interactions promote FliG ring formation on the cytoplasmic face of the MS ring. A conformational change in HelixMC connecting FliGM and FliGC is responsible for the switching between the counterclockwise (CCW) and clockwise (CW) rotational states of the FliG ring. However, it remains unknown how it occurs. Here, we carried out in vivo disulfide cross-linking experiments to see the effect of a CW-locked deletion (∆PAA) in FliG on the FliG ring structure in Salmonella enterica...
February 6, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Yukiko Cho, Takashi Ideue, Megumi Nagayama, Norie Araki, Tokio Tani
Satellite I RNA, a noncoding (nc)RNA transcribed from repetitive regions in human centromeres, binds to Aurora kinase B and forms a ncRNP complex required for chromosome segregation. To examine its function in this process, we purified satellite I ncRNP complex from nuclear extracts prepared from asynchronized or mitotic (M) phase-arrested HeLa cells and then carried out LC/MS to identify proteins bound to satellite I RNA. RBMX (RNA-binding motif protein, X-linked), which was isolated from M phase-arrested cells, was selected for further characterization...
January 31, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Jiyoung Lee, Ayumi Matsuzawa, Hirosuke Shiura, Akito Sutani, Fumitoshi Ishino
Epigenetic properties of cultured embryonic stem cells (ESCs), including DNA methylation imprinting, are important because they affect the developmental potential. Here, we tested a variety of culture media, including knockout serum replacement (KSR) and fetal bovine serum (FBS) with or without inhibitors of Gsk3β and Mek1/2 (2i) at various time points. In addition to the previously known passage-dependent global changes, unexpected dynamic DNA methylation changes occurred in both maternal and paternal differentially methylated regions: under the widely used condition of KSR with 2i, a highly hypomethylated state occurred at early passages (P1-7) as well as P10, but DNA methylation increased over further passages in most conditions, except under KSR with 2i at P25...
January 22, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Yasunobu Iwai, Keisuke Noda, Mizuho Yamazaki, Ayako Kato, Masaru Mezawa, Hideki Takai, Yohei Nakayama, Yorimasa Ogata
Follicular dendritic cell-secreted protein (FDC-SP) is a secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium. To elucidate the transcriptional regulation of the human FDC-SP gene by tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with chimeric constructs of the FDC-SP gene promoter linked to a luciferase reporter gene, gel mobility shift and chromatin immunoprecipitation assays using Ca9-22 gingival epithelial cells...
January 22, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Kazunori Watanabe, Ryuhei Yamaji, Takashi Ohtsuki
MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH-SY5Y cells. We identified 15 up-regulated and eight down-regulated miRNAs in SH-SY5Y cells treated with all-trans retinoic acid to induce differentiation...
January 17, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Bo Yang, Takahiro Kumoto, Takeshi Arima, Minako Nakamura, Yohei Sanada, Thanutchaporn Kumrungsee, Yusuke Sotomaru, Masayuki Shimada, Noriyuki Yanaka
To determine adipocytokines that play a regulatory role during obesity development, we explored the genes that encode growth factors and investigated the physiological functions for adipose tissue development. Here, we isolated amphiregulin (Areg) gene whose expression was significantly up-regulated in obese adipose tissues. Areg mRNA level was positively correlated with macrophage marker gene expression in adipose tissues in vivo. Unexpectedly, Areg transgenic mice showed less adipose tissue mass with increased mRNA expression levels of Tnf-α and peroxisome proliferator-activated receptor γ coactivator 1α (Pgc-1α) and delayed white adipose tissue development during the convalescent stage in a dextran sodium sulfate-induced colitis model...
January 17, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Makiko Ueda, Tetsuya Sato, Yasuyuki Ohkawa, Yoshihiro H Inoue
MicroRNAs (miRNAs) are involved in the regulation of important biological processes. Here, we describe a novel Drosophila miRNAs involved in aging. We selected eight Drosophila miRNAs, displaying high homology with seed sequences of aging-related miRNAs characterized in other species, and investigated whether the over-expression of these miRNAs affected aging in Drosophila adult flies. The lifespan of adults over-expressing miR-305, a miRNA showing high homology with miR-239 in C. elegans, was significantly shorter...
January 5, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Takuya Okada, Gaku Okabe, Yon-Soo Tak, Satoru Mimura, Haruhiko Takisawa, Yumiko Kubota
Intact G0 nuclei isolated from quiescent cells are not capable of DNA replication in interphase Xenopus egg extracts, which allow efficient replication of permeabilized G0 nuclei. Previous studies have shown multiple control mechanisms for maintaining the quiescent state, but DNA replication inhibition of intact G0 nuclei in the extracts remains poorly understood. Here, we showed that pre-RC is assembled on chromatin, but its activation is inhibited after incubating G0 nuclei isolated from quiescent NIH3T3 cells in the extracts...
January 4, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Kaori Yunoki, Sosuke Yoshinaga, Mitsuhiro Takeda, Ryohei Nagano, Yusuke Tsuchiya, Akihiro Sonoda, Tatsuichiro Tsuji, Makoto Hirakane, Etsuko Toda, Yuya Terashima, Kouji Matsushima, Hiroaki Terasawa
The control of protein solubility is a subject of broad interest. Although several solvent screening methods are available to search for compounds that enhance protein solubilization, their performance is influenced by the intrinsic solubility of the tested protein. We now present a method for screening solubilizing compounds, using an array of N- or C-terminal deletion mutants of the protein. A key behind this approach is that such terminal deletions of the protein affect its aggregation propensity. The solubilization activities of trial solvents are individually assessed, based on the number of solubilized mutants...
January 2, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Yasuhiro Hirano, Yasuha Kinugasa, Haruhiko Asakawa, Yuji Chikashige, Chikashi Obuse, Tokuko Haraguchi, Yasushi Hiraoka
Inner nuclear membrane (INM) proteins are thought to play important roles in modulating nuclear organization and function through their interactions with chromatin. However, these INM proteins share redundant functions in metazoans that pose difficulties for functional studies. The fission yeast Schizosaccharomyces pombe exhibits a relatively small number of INM proteins, and molecular genetic tools are available to separate their redundant functions. In S. pombe, it has been reported that among potentially redundant INM proteins, Lem2 displays a unique genetic interaction with another INM protein, Bqt4, which is involved in anchoring telomeres to the nuclear envelope...
January 2, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Natsuko Kinoshita, Catalina Arenas-Huertero, Nam-Hai Chua
By modifying the existing cytosolic RNA visualization tool pioneered by Schönberger, Hammes, and Dresselhaus (2012), we developed a method to visualize nuclear-localized RNA. Our method uses (i) an RNA component that consists of an RNA of interest that is fused to a bacteriophage-derived MS2 sequence; and (ii) GFP fused to MS2 coat protein (MSCP), which binds specifically to MS2 as is also the case in the method for cytosolic RNA visualization. The nuclear localization sequence (NLS) at the C-terminal of MSCP-GFP tethers the probe to the nucleus...
December 22, 2017: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Osamu Kaminuma, Shigeki Katoh, Taeko Miyagi, Nobumasa Watanabe, Noriko Kitamura, Tomoe Nishimura, Mayumi Saeki, Akio Mori, Takachika Hiroi
Neuraminidase family enzymes that hydrolyze the terminal sialic acid linkage in biomolecules are involved in various immune responses. We previously showed that Th1 and Th2 cells differentially express several neuraminidases. Herein, the expression of neuraminidases in induced regulatory T (iTreg) cells was investigated in comparison with that in other T-cell subsets. Contrary to the tendency toward higher neuraminidase 1 mRNA expression in in vitro-differentiated Th2 cells, compared to Th1, Th17 and iTreg cells, we observed significantly higher expression of neuraminidase 3 (Neu3) in iTreg cells...
December 22, 2017: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Munehiro Ohashi, Yasuhiro Umemura, Nobuya Koike, Yoshiki Tsuchiya, Yutaka Inada, Hitomi Watanabe, Tomoko Tanaka, Yoichi Minami, Osamu Ukimura, Tsuneharu Miki, Tatsuro Tajiri, Gen Kondoh, Yasuhiro Yamada, Kazuhiro Yagita
The circadian clock, which regulates cellular physiology, such as energy metabolism, resides in each cell level throughout the body. Recently, it has been elucidated that the cellular circadian clock is closely linked with cellular differentiation. Moreover, the misregulation of cellular differentiation in mouse embryonic stem cells (ESCs) induced abnormally differentiated cells with impaired circadian clock oscillation, concomitant with the post-transcriptional suppression of CLOCK proteins. Here, we show that the circadian molecular oscillation is disrupted in dysdifferentiation-mediated mouse kidney tumors induced by partial in vivo reprogramming, resembling Wilms tumors...
December 22, 2017: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
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