Masashi Yukawa

Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases.

Human mitochondrial (mt) tRNALys has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (τm5s2U), at its anticodon wobble position. We previously found that the mt tRNALys, carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the τm5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.

Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice

Histone H2A has several variants, and changes in chromatin composition associated with their replacement might involve chromatin structure remodeling. We examined the dynamics of the canonical histone H2A and its three variants, H2A.X, H2A.Z and macroH2A, in the mouse during oogenesis and pre-implantation development when genome remodeling occurs. Immunocytochemistry with specific antibodies revealed that, although H2A and all variants were deposited in the nuclei of full-grown oocytes, only histone H2A.X was abundant in the pronuclei of one-cell embryos after fertilization, in contrast with the low abundance of histone H2A and the absence of H2A.Z. The decline in H2A and the depletion of H2A.Z and macroH2A after fertilization were confirmed using Flag epitope-tagged H2A, H2A.Z and macroH2A transgenic mouse lines. Microinjection experiments with mRNA encoding the Flag-tagged proteins revealed a similar pattern of nuclear incorporation of the H2A variants. Fusion protein experiments using H2A, H2A.Z and macroH2A fused with the C-terminal 23 amino acids of H2A.X showed that the C-terminal amino acids of H2A.X function specifically to target this variant histone into chromatin in embryos after fertilization and that the absence of H2A.Z and macroH2A from the chromatin is required for normal development. These results suggest that global changes in the composition of histone H2A variants in chromatin play a role in genome remodeling after fertilization.

Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae

Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.

Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

A microtubule nucleator γ-tubulin forms a multiprotein complex (γ-tubulin complex or γ-TuC), which localizes to the microtubule organizing centers (MTOCs). We identify fission yeast Mzt1/MOZART1 as a novel conserved stoichiometric component of the γ-TuC. Mzt1 is required for cell viability, microtubule organization, and γ-TuC localization to the MTOCs, yet the core γ-TuC assembles in its absence.

Interplay between Chromatin and trans-Acting Factors on the IME2 Promoter upon Induction of the Gene at the Onset of Meiosis

ABSTRACT The IME2 gene is one of the key regulators of the initiation of meiosis in budding yeast. This gene is repressed during mitosis through the repressive chromatin structure at the promoter, which is maintained by the Rpd3-Sin3 histone deacetylase (HDAC) complex. IME2 expression in meiosis requires Gcn5/histone acetyltransferase, the transcriptional activator Ime1, and the chromatin remodeler RSC; however, the molecular basis of IME2 activation had not been previously defined. We found that, during mitotic growth, a nucleosome masked the TATA element of IME2, and this positioning depended on HDAC. This chromatin structure was remodeled at meiosis by RSC that was recruited to TATA by Ime1. Stable tethering of Ime1 to the promoter required the presence of Gcn5. Interestingly, Ime1 binding to the promoter was kept at low levels during the very early stages in meiosis, even when the levels of Ime1 and histone H3 acetylation at the promoter were at their highest, making a 4- to 6-h delay of the IME2 expression from that of IME1. HDAC was continuously present at the promoter regardless of the transcriptional condition of IME2, and deletion of RPD3 allowed the IME2 expression shortly after the expression of IME1, suggesting that HDAC plays a role in regulating the timing of IME2 expression.

Nps1/Sth1p, a component of an essential chromatin‐remodeling complex of Saccharomyces cerevisiae, is required for the maximal expression of early meiotic genes

The NPS1/STH1 gene of Saccharomyces cerevisiae is essential for mitotic growth, especially for the progression through the G2/M phase. It encodes a major component of the chromatin‐remodelling complex, RSC, of unknown function. We attempted to address the function of NPS1 in meiosis.

Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate‐induced calcium oscillation, even in growth factor‐free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor α (TGFα), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.

Data mining tools for the Saccharomyces cerevisiae morphological database

For comprehensive understanding of precise morphological changes resulting from loss-of-function mutagenesis, a large collection of 1 899 247 cell images was assembled from 91 271 micrographs of 4782 budding yeast disruptants of non-lethal genes. All the cell images were processed computationally to measure ∼500 morphological parameters in individual mutants. We have recently made this morphological quantitative data available to the public through the Saccharomyces cerevisiae Morphological Database (SCMD). Inspecting the significance of morphological discrepancies between the wild type and the mutants is expected to provide clues to uncover genes that are relevant to the biological processes producing a particular morphology. To facilitate such intensive data mining, a suite of new software tools for visualizing parameter value distributions was developed to present mutants with significant changes in easily understandable forms. In addition, for a given group of mutants associated with a particular function, the system automatically identifies a combination of multiple morphological parameters that discriminates a mutant group from others significantly, thereby characterizing the function effectively. These data mining functions are available through the World Wide Web at .

The Msd1-Wdr8-Pkl1 complex anchors microtubule minus ends to fission yeast spindle pole bodies.

Msd1–Wdr8 are delivered by Pkl1 to mitotic spindle pole bodies, where the Msd1–Wdr8–Pkl1 complex anchors the minus ends of spindle microtubules and antagonizes the outward-pushing forces generated by Cut7/kinesin-5 in fission yeast.

An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation

ABSTRACT Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3–Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module. Summary: We show that Dis1 and Mal3, fission yeast XMAP215/TOG and EB1 family members, directly interact in a non-canonical manner, thereby synergistically enhancing microtubule dynamics.