The mitochondrial RNA landscape of Saccharomyces cerevisiae.

Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this in silico approach, the essential RNase of the nuclear and cytoplasmic exosome, Dis3p, emerges as a strong candidate. Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay.

The ribonuclease Dis3 is an essential regulator of the developmental transcriptome.

BACKGROUND: Dis3 is ribonuclease that acts directly in the processing, turnover, and surveillance of a large number of distinct RNA species. Evolutionarily conserved from eubacteria to eukaryotes and a crucial component of the RNA processing exosome, Dis3 has been shown to be essential in yeast and fly S2 cells. However, it is not known whether Dis3 has essential functions in a metazoan. This study inquires whether Dis3 is required for Drosophila development and viability and how Dis3 regulates the transcriptome in the developing fly. RESULTS: Using transgenic flies, we show that Dis3 knock down (Dis3KD) retards growth, induces melanotic tumor formation, and ultimately results in 2nd instar larval lethality. In order to determine whether Dis3KD fly phenotypes were a consequence of disrupting developmentally regulated RNA turnover, we performed RNA deep sequencing analysis on total RNA isolated from developmentally staged animals. Bioinformatic analysis of transcripts from Dis3KD flies reveals substantial transcriptomic changes, most notably down-regulation in early expressed RNAs. Finally, gene ontology analysis of this early stage shows that Dis3 regulates transcripts related to extracellular structure and remodelling, neurogenesis, and nucleotide metabolism. CONCLUSIONS: We conclude that Dis3 is essential for early Drosophila melanogaster development and has specific and important stage-specific roles in regulating RNA metabolism. In showing for the first time that Dis3 is required for the development of a multicellular organism, our work provides mechanistic insight into how Dis3-either independent of or associated with the RNA processing exosome-participates in cell type-specific RNA turnover in metazoan development.

Dis3- and exosome subunit-responsive 3' mRNA instability elements.

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.

Pronounced and extensive microtubule defects in a Saccharomyces cerevisiae DIS3 mutant.

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.

Theory of the origin, evolution, and nature of life.

Life is an inordinately complex unsolved puzzle. Despite significant theoretical progress, experimental anomalies, paradoxes, and enigmas have revealed paradigmatic limitations. Thus, the advancement of scientific understanding requires new models that resolve fundamental problems. Here, I present a theoretical framework that economically fits evidence accumulated from examinations of life. This theory is based upon a straightforward and non-mathematical core model and proposes unique yet empirically consistent explanations for major phenomena including, but not limited to, quantum gravity, phase transitions of water, why living systems are predominantly CHNOPS (carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur), homochirality of sugars and amino acids, homeoviscous adaptation, triplet code, and DNA mutations. The theoretical framework unifies the macrocosmic and microcosmic realms, validates predicted laws of nature, and solves the puzzle of the origin and evolution of cellular life in the universe.

The exozyme model: a continuum of functionally distinct complexes.

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.

Drosophila melanogaster Dis3 N-terminal domains are required for ribonuclease activities, nuclear localization and exosome interactions.

Eukaryotic cells use numerous pathways to regulate RNA production, localization and stability. Several of these pathways are controlled by ribonucleases. The essential ribonuclease, Dis3, plays important roles in distinct RNA metabolic pathways. Despite much progress in understanding general characteristics of the Dis3 enzyme in vitro and in vivo, much less is known about the contributions of Dis3 domains to its activities, subcellular localization and protein-protein interactions. To address these gaps, we constructed a set of Drosophila melanogaster Dis3 (dDis3) mutants and assessed their enzymatic activity in vitro and their localizations and interactions in S2 tissue culture cells. We show that the dDis3 N-terminus is sufficient for endoribonuclease activity in vitro and that proper N-terminal domain structure is critical for activity of the full-length polypeptide. We find that the dDis3 N-terminus also contributes to its subcellular distribution, and is necessary and sufficient for interactions with core exosome proteins. Finally, dDis3 interaction with dRrp6 and dImportin-α3 is independent of core interactions and occurs though two different regions. Taken together, our data suggest that the dDis3 N-terminus is a dynamic and complex hub for RNA metabolism and exosome interactions.

Genome-wide analysis reveals distinct substrate specificities of Rrp6, Dis3, and core exosome subunits.

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.

Characterization of the Drosophila melanogaster Dis3 ribonuclease.

The Dis3 ribonuclease is a member of the hydrolytic RNR protein family. Although much progress has been made in understanding the structure, function, and enzymatic activities of prokaryotic RNR family members RNase II and RNase R, there are no activity studies of the metazoan ortholog, Dis3. Here, we characterize the activity of the Drosophila melanogaster Dis3 (dDis3) protein. We find that dDis3 is active in the presence of various monovalent and divalent cations, and requires divalent cations for activity. dDis3 hydrolyzes compositionally distinct RNA substrates, yet releases different products depending upon the substrate. Additionally, dDis3 remains active when lacking N-terminal domains, suggesting that an independent active site resides in the C-terminus of the protein. Finally, a study of dDis3 interactions with dRrp6 and core exosome subunits in extracts revealed sensitivity to higher divalent cation concentrations and detergent, suggesting the presence of both ionic and hydrophobic interactions in dDis3-exosome complexes. Our study thus broadens our mechanistic understanding of the general ribonuclease activity of Dis3 and RNR family members.

Core exosome-independent roles for Rrp6 in cell cycle progression.

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.

Interdependent nucleocytoplasmic trafficking and interactions of Dis3 with Rrp6, the core exosome and importin-alpha3.

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome-binding protein Dis3, a processive 3' to 5' exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N-terminal domain mutants of dDis3 abolish associations with the 'core' exosome, yet only reduce binding to the 'nuclear' exosome-associated factor dRrp6. We show that nuclear localization of dDis3 requires a C-terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co-precipitates the NLS-binding protein importin-alpha3. Surprisingly, dDis3 constructs that lack or mutate the C-terminal NLS retain importin-alpha3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin-alpha3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.

Immobility, inheritance and plasticity of shape of the yeast nucleus.

BACKGROUND: Since S. cerevisiae undergoes closed mitosis, the nuclear envelope of the daughter nucleus is continuous with that of the maternal nucleus at anaphase. Nevertheless, several constitutents of the maternal nucleus are not present in the daughter nucleus. The present study aims to identify proteins which impact the shape of the yeast nucleus and to learn whether modifications of shape are passed on to the next mitotic generation. The Esc1p protein of S. cerevisiae localizes to the periphery of the nucleoplasm, can anchor chromatin, and has been implicated in targeted silencing both at telomeres and at HMR. RESULTS: Upon increased Esc1p expression, cell division continues and dramatic elaborations of the nuclear envelope extend into the cytoplasm. These "escapades" include nuclear pores and associate with the nucleolus, but exclude chromatin. Escapades are not inherited by daughter nuclei. This exclusion reflects their relative immobility, which we document in studies of prezygotes. Moreover, excess Esc1p affects the levels of multiple transcripts, not all of which originate at telomere-proximal loci. Unlike Esc1p and the colocalizing protein, Mlp1p, overexpression of selected proteins of the inner nuclear membrane is toxic. CONCLUSION: Esc1p is the first non-membrane protein of the nuclear periphery which - like proteins of the nuclear lamina of higher eukaryotes - can modify the shape of the yeast nucleus. The elaborations of the nuclear envelope ("escapades") which appear upon induction of excess Esc1p are not inherited during mitotic growth. The lack of inheritance of such components could help sustain cell growth when parental nuclei have acquired potentially deleterious characteristics.

Differential distribution of exosome subunits at the nuclear lamina and in cytoplasmic foci.

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.

Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. Here, we report that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, we discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction.

One-hybrid screens at the Saccharomyces cerevisiae HMR locus identify novel transcriptional silencing factors.

In Saccharomyces cerevisiae, genes located at the telomeres and the HM loci are subject to transcriptional silencing. Here, we report results of screening a Gal4 DNA-binding domain hybrid library for proteins that cause silencing when targeted to a silencer-defective HMR locus.

Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo.

RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). Here we show that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III-transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. Our observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms.

The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila.

The RNA polymerase II elongation complex contains several factors that facilitate transcription elongation and catalyse the processing of precursor messenger RNAs (pre-mRNAs). The conserved elongation factor Spt6 is recruited rapidly and robustly to sites of active transcription. Here we show that Drosophila Spt6 (dSpt6) co-purifies with the exosome, a complex of 3' to 5' exoribonucleases that is implicated in the processing of structural RNA and in the degradation of improperly processed pre-mRNA. Immunoprecipitation assays of Drosophila nuclear extracts show that the exosome also associates with the elongation factor dSpt5 and RNA polymerase II. In vivo, exosome subunits colocalize with dSpt6 at transcriptionally active loci on polytene chromosomes during normal development and are strongly recruited to heat-shock loci on gene induction. At higher resolution, chromatin immunoprecipitation analysis shows that the exosome is recruited to transcriptionally active units of heat-shock genes. These data provide a physical basis for the hypothesis that exosome-mediated pre-mRNA surveillance accompanies transcription elongation.

Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning.

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.