We present an analysis of atmospheric neutrino data from a 33.0 kton yr (535-day) exposure of the Super-Kamiokande detector. The data exhibit a zenith angle dependent deficit of muon neutrinos which is inconsistent with expectations based on calculations of the atmospheric neutrino flux. Experimental biases and uncertainties in the prediction of neutrino fluxes and cross sections are unable to explain our observation. The data are consistent, however, with two-flavor n m $ n t oscillations with sin 2 2u . Atmospheric neutrinos are produced as decay products in hadronic showers resulting from collisions of cosmic rays with nuclei in the upper atmosphere. Production of electron and muon neutrinos is dominated by the processes p 1 ! m 1 1 n m followed by m 1 ! e 1 1 n m 1 n e (and their charge conjugates) giving an expected ratio 1562 0031-9007͞98͞81(8)͞1562(6)$15.00
CRM1 is distantly related to receptors that mediate nuclear protein import and was previously shown to interact with the nuclear pore complex. Overexpression of CRM1 in Xenopus oocytes stimulates Rev and U snRNA export from the nucleus. Conversely, leptomycin B, a cytotoxin that is shown to bind to CRM1 protein, specifically inhibits the nuclear export of Rev and U snRNAs. In vitro, CRM1 forms a leptomycin B-sensitive complex involving cooperative binding of both RanGTP and the nuclear export signal (NES) from either the Rev or PKI proteins. We conclude that CRM1 is an export receptor for leucine-rich nuclear export signals and discuss a model for the role of RanGTP in CRM1 function and in nuclear export in general.
Reversible acetylation of alpha-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated alpha-tubulin is tightly controlled and stereotypic. Acetylated alpha-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it associates with microtubules and localizes with the microtubule motor complex containing p150(glued) (ref. 3). In vivo, the overexpression of HDAC6 leads to a global deacetylation of alpha-tubulin, whereas a decrease in HDAC6 increases alpha-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates alpha-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biological processes beyond histone metabolism and gene transcription.
SUMMARYHistone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc-or NAD + -dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases.
The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.
The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB. 1 H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michaeltype addition, consistent with the idea that LMB binds covalently via its ␣,␤-unsaturated ␦-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.Many cellular proteins either reside in the nucleus or shuttle between the nucleus and the cytoplasm by energy-dependent transport across the nuclear envelope. Specific sequences within a protein contain the information necessary for the nucleocytoplasmic transport: most nuclear proteins have nuclear localization sequences (NLS) rich in basic amino acids, whereas others carry short nuclear export sequences (NES) rich in leucine (1, 2). CRM1͞exportin 1 was shown to be a receptor for the NES in both lower and higher eukaryotes (3-6). Genetic alterations in the CRM1 locus caused a defect in nuclear export of NES-bearing proteins in yeast (3,5,7,8). Nuclear microinjection of a CRM1-specific antibody that prevents the in vitro NES binding inhibited in vivo protein nuclear export in mammalian cells (9). Thus, the NESmediated nuclear export of proteins is a universal and conserved mechanism by which subcellular localization of proteins is controlled in cells.CRM1 originally was identified as a protein essential for maintaining chromosome structure in the fission yeast Schizosaccharomyces pombe (10). The functional homologues that complement the fission yeast crm1 mutation were cloned from the budding yeast Saccharomyces cerevisiae (11) and from human cells (8,12). We showed previously that a mutation (crm1-N1) of S. pombe crm1 ϩ conferred resistance to leptomycin B (LMB) (13), which had been discovered as a potent antifungal antibiotic blocking the eukaryotic cell cycle (14, 15). In contrast, the cold-sensitive crm1-809 mutant strain was hypersensitive to LMB. Furthermore, morphological and biochemical phenotypes of crm1-809 mutant cells at nonpermissive temperature ...
The molecular chaperone heat shock protein 90 (Hsp90) and its accessory cochaperones function by facilitating the structural maturation and complex assembly of client proteins, including steroid hormone receptors and selected kinases. By promoting the activity and stability of these signaling proteins, Hsp90 has emerged as a critical modulator in cell signaling. Here, we present evidence that Hsp90 chaperone activity is regulated by reversible acetylation and controlled by the deacetylase HDAC6. We show that HDAC6 functions as an Hsp90 deacetylase. Inactivation of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from an essential cochaperone, p23, and a loss of chaperone activity. In HDAC6-deficient cells, Hsp90-dependent maturation of the glucocorticoid receptor (GR) is compromised, resulting in GR defective in ligand binding, nuclear translocation, and transcriptional activation. Our results identify Hsp90 as a target of HDAC6 and suggest reversible acetylation as a unique mechanism that regulates Hsp90 chaperone complex activity.
The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.
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