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ABSTRACT: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3'-5' exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3' untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA turnover that involves direct Dis3 and other exosome subunit recruitment to and/or regulation on mRNA substrates.
Biochemical and Biophysical Research Communications 06/2012; 423(3):461-6. · 2.48 Impact Factor
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ABSTRACT: Subunits of the RNA processing exosome assemble into structurally distinct protein complexes that function in disparate cellular compartments and RNA metabolic pathways. Here, in a genetic, cell biological and transcriptomic analysis, we examined the role of Dis3, an essential polypeptide with endo- and 3'→5' exo-ribonuclease activity, in cell cycle progression. We present several lines of evidence that perturbation of DIS3 affects microtubule (MT) localization and structure in Saccharomyces cerevisiae. Cells with a DIS3 mutant: (a) accumulate anaphase and pre-anaphase mitotic spindles; (b) exhibit spindles that are misorientated and displaced from the bud neck; (c) harbour elongated spindle-associated astral MTs; (d) have an increased G1 astral MT length and number; and (e) are hypersensitive to MT poisons. Mutations in the core exosome genes RRP4 and MTR3 and the exosome cofactor gene MTR4, but not other exosome subunit gene mutants, also elicit MT phenotypes. RNA deep sequencing analysis (RNA-seq) shows broad changes in the levels of cell cycle- and MT-related transcripts in mutant strains. Collectively, the data presented in this study suggest an evolutionarily conserved role for Dis3 in linking RNA metabolism, MTs and cell cycle progression.
Yeast 07/2011; 28(11):755-69. · 1.89 Impact Factor
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ABSTRACT: Exosome complexes are composed of 10 to 11 subunits and are involved in multiple facets of 3' → 5' RNA processing and turnover. The current paradigm stipulates that a uniform, stoichiometric core exosome, composed of single copies of each subunit, carries out all RNA metabolic functions in vivo. While core composition is well established in vitro, available genetic, cell biological, proteomic, and transcriptomic data raise questions about whether individual subunits contribute to RNA metabolic functions exclusively within the complex. Here, we recount the current understanding of the core exosome model and show predictions of the core model that are not satisfied by the available evidence. To resolve this discrepancy, we propose the exozyme hypothesis, a novel model stipulating that while exosome subunits can and do carry out certain functions within the core, subsets of exosome subunits and cofactors also assemble into a continuum of compositionally distinct complexes--exozymes--with different RNA specificities. The exozyme model is consistent with all published data and provides a new framework for understanding the general mechanisms and regulation of RNA processing and turnover.
RNA 11/2010; 17(1):1-13. · 5.09 Impact Factor
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ABSTRACT: The RNA processing exosome complex was originally defined as an evolutionarily conserved multisubunit complex of ribonucleases responsible for the processing and/or turnover of stable RNAs. The exosome complex is also involved in the surveillance of mRNAs in both the nucleus and the cytoplasm, including nonsense-mediated decay (NMD) targets. The detailed mechanisms for how individual exosome subunits participate in each of these RNA metabolic pathways remains unclear. Here, we use RNAi to deplete exosome subunits, the exonucleases Rrp6 and Dis3, and an exosome cofactor in Drosophila melanogaster S2 tissue culture cells and assay the effects on global mRNA levels using gene expression microarrays. Consistent with the RNA degradative activities ascribed to the exosome, most mRNAs are increased. Notably, these stabilized mRNAs possess 3' untranslated regions that are longer than the representative transcriptomic average. Moreover, our results reveal substantial differences in the pools of affected mRNAs for each depleted subunit. For example, approximately 25% of the affected transcripts in Rrp6 depleted cells represent NMD substrates. While the affected mRNAs were dissimilar, they encode proteins that function in similar cellular pathways. We conclude that individual exosome subunits are largely functionally independent at the transcript level, but are interdependent on a transcriptomic level.
RNA 02/2010; 16(4):781-91. · 5.09 Impact Factor
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ABSTRACT: Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are key epigenetic regulators of global transcription programs. Their antagonistic chromatin-modifying activities modulate the expression of many genes and affect many biological processes. Here we report that heterozygous mutations in two core subunits of Polycomb Repressive Complex 2 (PRC2), the histone H3 lysine 27 (H3K27)-specific methyltransferase E(Z) and its partner, the H3 binding protein ESC, increase longevity and reduce adult levels of trimethylated H3K27 (H3K27me3). Mutations in trithorax (trx), a well known antagonist of Polycomb silencing, elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. Like many long-lived mutants, E(z) and esc mutants exhibit increased resistance to oxidative stress and starvation, and these phenotypes are also suppressed by trx mutations. This suppression strongly suggests that both the longevity and stress resistance phenotypes of PRC2 mutants are specifically due to their reduced levels of H3K27me3 and the consequent perturbation of Polycomb silencing. Consistent with this, long-lived E(z) mutants exhibit derepression of Abd-B, a well-characterized direct target of Polycomb silencing, and Odc1, a putative direct target implicated in stress resistance. These findings establish a role for PRC2 and TRX in the modulation of organismal longevity and stress resistance and indicate that moderate perturbation of Polycomb silencing can increase longevity.
Proceedings of the National Academy of Sciences 12/2009; 107(1):169-74. · 9.68 Impact Factor
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ABSTRACT: Exosome complexes are 3' to 5' exoribonucleases composed of subunits that are critical for numerous distinct RNA metabolic (ribonucleometabolic) pathways. Several studies have implicated the exosome subunits Rrp6 and Dis3 in chromosome segregation and cell division but the functional relevance of these findings remains unclear. Here, we report that, in Drosophila melanogaster S2 tissue culture cells, dRrp6 is required for cell proliferation and error-free mitosis, but the core exosome subunit Rrp40 is not. Micorarray analysis of dRrp6-depleted cell reveals increased levels of cell cycle- and mitosis-related transcripts. Depletion of dRrp6 elicits a decrease in the frequency of mitotic cells and in the mitotic marker phospho-histone H3 (pH3), with a concomitant increase in defects in chromosome congression, separation, and segregation. Endogenous dRrp6 dynamically redistributes during mitosis, accumulating predominantly but not exclusively on the condensed chromosomes. In contrast, core subunits localize predominantly to MTs throughout cell division. Finally, dRrp6-depleted cells treated with microtubule poisons exhibit normal kinetochore recruitment of the spindle assembly checkpoint protein BubR1 without restoring pH3 levels, suggesting that these cells undergo premature chromosome condensation. Collectively, these data support the idea that dRrp6 has a core exosome-independent role in cell cycle and mitotic progression.
Molecular biology of the cell 03/2009; 20(8):2242-53. · 5.98 Impact Factor
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ABSTRACT: The exosome complex plays important roles in RNA processing and turnover. Despite significant mechanistic insight into exosome function, we still lack a basic understanding of the subcellular locales where exosome complex biogenesis and function occurs. Here, we employ a panel of Drosophila S2 stable cell lines expressing epitope-tagged exosome subunits to examine the subcellular distribution of exosome complex components. We show that tagged Drosophila exosome subunits incorporate into complexes that recover endogenous nuclear and cytoplasmic exosome subunits. Immunolocalization analyses demonstrate that subsets of both epitope-tagged and endogenous exosome subunits are enriched in discrete subcellular compartments. In particular, dRrp4, dRrp42, dRrp46, and dCsl4 are enriched in cytoplasmic foci. Although dRrp4 and dRrp42 sometimes colocalize with dCsl4, these subunits are predominantly found in distinct cytoplasmic compartments. Strikingly, dRrp44/dDis3 and dRrp41/dSki6 colocalize with the nuclear lamina and often exhibit a restricted and asymmetric distribution at the nuclear periphery. Taken together, these observations indicate that individual exosome subunits have distinct localizations in vivo. These different distribution patterns presumably reflect distinct exosome subunit subcomplexes with correspondingly specialized functions.
Molecular Biology of the Cell 04/2006; 17(3):1399-409. · 4.94 Impact Factor
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ABSTRACT: The distal serpin subcluster contains genes encoding alpha1-antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL-like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue-specific expression of these genes associates with astrocyte- and hepatocyte-specific chromatin structures. In hepatocytes, we identified 12 Dnase I-hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5' flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue-specific expression pattern of these serpin genes.
Journal of Neurochemistry 09/2005; 94(3):763-73. · 4.06 Impact Factor
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ABSTRACT: Expression of alpha1antichymotrypsin (ACT) is significantly activated by interleukin-1 (IL-1) in human astrocytes; however, it is barely affected by IL-1 in hepatocytes. This tissue-specific regulation depends upon an enhancer that contains both nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) elements, and is also observed for an NF-kappaB reporter but not for an AP-1 reporter. We found efficient activation of NF-kappaB binding in both cell types; however, this binding was persistent in glial cells and only transient in hepatocytes. IL-1-activated NF-kappaB complexes consisted of p65 and p50, with p65 transiently phosphorylated on serine 536 in glial cells whereas more persistently in hepatic cells. Overexpression of p65 or constitutively active IKKbeta (inhibitor of NF-kappaB kinase beta) resulted in an efficient activation of the ACT reporter in hepatic cells, indicating that a specific mechanism exists in these cells terminating IL-1 signaling. IL-1 effectively induced the degradation of inhibitor of NF-kappaBalpha (IkBalpha) and IkBepsilon in both cell types but IkBbeta was not affected. However, IkBalpha was resynthesized much more rapidly in hepatic cells in comparison to glial cells. In addition, the initial levels of IkBalpha were much lower in glial cells. We propose that the tissue-specific regulation of the ACT gene expression by IL-1 is determined by different efficiencies of IkBalpha resynthesis in glial and hepatic cells.
Journal of Neurochemistry 03/2005; 92(4):730-8. · 4.06 Impact Factor
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ABSTRACT: Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the -58 to -51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that down-regulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor-kappaB (IkappaB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. We conclude that OSM and IL-1 regulate the PAI-1 gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the PAI-1 gene.
Journal of Neurochemistry 12/2002; 83(3):696-703. · 4.06 Impact Factor
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ABSTRACT: Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that down-regulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor-κB (IκB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. We conclude that OSM and IL-1 regulate the PAI-1 gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the PAI-1 gene.
Journal of Neurochemistry 10/2002; 83(3):696 - 703. · 4.06 Impact Factor
