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PubMed2006

Contribution of Trf4/5 and the nuclear exosome to genome stability through regulation of histone mRNA levels in Saccharomyces cerevisiae.

Balanced levels of histones are crucial for chromosome stability, and one major component of this control regulates histone mRNA amounts. The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in a quality control mechanism that mediates polyadenylation and consequent degradation of various RNA species by the nuclear exosome. None of the known RNA targets, however, explains the fact that trf mutants have specific cell cycle defects consistent with a role in maintaining genome stability. Here, we investigate the role of Trf4/5 in regulation of histone mRNA levels. We show that loss of Trf4 and Trf5, or of Rrp6, a component of the nuclear exosome, results in elevated levels of transcripts encoding DNA replication-dependent histones. Suggesting that increased histone levels account for the phenotypes of trf mutants, we find that TRF4 shows synthetic genetic interactions with genes that negatively regulate histone levels, including RAD53. Moreover, synthetic lethality of trf4Delta rad53Delta is rescued by reducing histone levels whereas overproduction of histones is deleterious to trf's and rrp6Delta mutants. These results identify TRF4, TRF5, and RRP6 as new players in the regulation of histone mRNA levels in yeast. To our knowledge, the histone transcripts are the first mRNAs that are upregulated in Trf mutants.

Blotting, Northern↗
PubMed2006

[Production of specific CTL induced by exosomes derived from K562 cells].

The aim of this study was to investigate whether exosomes derived from K562 cells and human monocyte-derived dendritic cells (DCs) transfected with total RNA of K562 cells are capable of inducing antigen-specific cytotoxic T lymphocytes (CTL) responses in vitro. DCs were generated from peripheral blood mononuclear cells (PBMNC) of healthy volunteers in the presence of GM-CSF and IL-4, and then were transfected with K562 RNA by using DOTAP lipofection. Exosomes was extracted from the supernatant of DCs and K562 cells. The T cell were activated to be tumor specific CTL after DCs and exosomes were co-cultured with autologous T cells derived from healthy volunteers' PBMNC. The effect of CTL on K562 cells was detected by MTT assay. The results showed that treatment of T cells with exosomes derived from K562 cells or DCs transfected with total RNA of K562 cells could significantly promote their killing ability on K562 cells as compared with untreated T cells (P < 0.05). The killing ability of T cells treated with exosomes on K562 cells was stronger than on HL-60 cells (P < 0.05). It is concluded that the specific CTL immune response to leukemia cells can be induced by exosomes derived from K562 cells.

Dendritic Cells↗
PubMed2003

Contribution of domain structure to the RNA 3' end processing and degradation functions of the nuclear exosome subunit Rrp6p.

The 3'-5' riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3' ends of 5.8S rRNA, sno- and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3' ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3'-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.

Amino Acid Sequence↗
PubMed2004

Autoantibodies to protein transport and messenger RNA processing pathways: endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies.

Over 50 years ago the lupus erythematosus (LE) cell phenomenon was described and this was quickly followed by the introduction of the LE cell test and indirect immunofluorescence (IIF) to detect antinuclear antibodies (ANA) in clinical laboratories. Recently, attention has turned to the identification of the autoantigens that bind to cytoplasmic organelles such as the Golgi complex, endosomes and other "cytoplasmic somes". Three endosome autoantigens include early endosome antigen 1 (EEA1, 160 kDa), cytoplasmic linker protein-170 (CLIP-170, 170 kDa), and lysobisphosphatidic acid (LBPA). Antibodies to EEA1 were seen in a variety of conditions but approximately 40% of the patients had a neurological disease. Despite the prominence of lysosomes in cells and tissues, reports of autoantibodies are limited to the lysosomal antigen h-LAMP-2 and the cytoplasmic antineutrophil antibodies (cANCA). Autoantigens in the Golgi complex include giantin/macrogolgin, golgin-245, golgin 160, golgin-97, golgin 95/gm130, and golgin-67. More recently, there has been an interest in autoantibodies that bind components of the "SMN complex" or the "assemblyosome". Arginine/glycine (RG)-rich domains in components of the SMN complex interact with Sm, like-Sm (LSm), fibrillarin, RNA helicase A (Gu), and coilin proteins, all of which are antigen targets in a variety of diseases. More recently, components of a novel cytoplasmic structure named GW bodies (GWBs) have been identified as targets of human autoantibodies. Components of GWBs include GW182, a unique mRNA-binding protein, like Sm proteins (LSms), and decapping (hDcp1) and exonuclease (Xrn) enzymes. Current evidence suggests that GWBs are involved in the cytoplasmic processing of mRNAs. Autoantibodies to the "cytoplasmic somes" are relatively uncommon and serological tests to detect most of them are not widely available.

Animals↗
PubMed2005

Reviving the exosome.

In the nucleus of eukaryotic cells, RNA decay in the 3'-5' direction is carried out by a complex of exonucleases called the exosome. Surprisingly, the purified exosome shows only weak activity in vitro. Two papers in this issue of Cell, by LaCava et al. (2005) and Wyers et al. (2005), and a third report by Vanacova et al. (2005) help to solve this riddle by characterizing a new RNA decay-activating complex containing a poly(A) polymerase. In addition, they identify new unconventional RNA polymerase II transcription units.

Cell Nucleus↗
PubMed2006

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.

Animals↗
PubMed2006

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex.

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.

Catalysis↗
PubMed2000

The human exosome: an autoantigenic complex of exoribonucleases in myositis and scleroderma.

The anti-PM/Scl autoantibodies are known to characterize a subset of autoimmune patients with myositis, scleroderma (Scl), and the PM/Scl overlap syndrome. The major autoantigens that are recognized by anti-PM/Scl autoantibodies are designated PM/Scl-100 and PM/Scl-75. These autoantigens have been reported to associate into a large complex consisting of 11 to 16 proteins and to play a role in ribosome synthesis. Recently, it was discovered that the PM/Scl complex is the human counterpart of the yeast (Saccharomyces cerevisiae) exosome, which is an RNA-processing complex consisting of 11 3' --> 5' exoribonucleases. To date, 10 human exosome components have been identified, although only some of these were studied in more detail. In this review, we discuss some recent advances in the characterization of the PM/Scl complex.

Autoantigens↗
PubMed2025

LncRNA Profiling and ceRNA Network Construction of Intrauterine Exosomes in Goats During Embryo Implantation.

Exosomes have been shown to play an important role in embryo implantation, but the mechanism is still unclear. This study aimed to investigate the functional roles of lncRNAs in intrauterine exosomes in goat pregnancy. We used RNA-seq to identify the lncRNA profiles of exosomes obtained from goat uterine rinsing fluid at 5, 15, and 18 days of gestation. In addition, we performed weighted gene co-expression network analysis based on differentially expressed mRNAs (DEMs) and lncRNAs (DELs). Functional enrichment analyses of gene modules were conducted using Gene Ontology classification (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. A lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory network was constructed based on predictive interaction derived from miRTarBase, miRDB and RNAhybrid databases. Altogether, 831 DELs were identified. GO and KEGG analysis showed that the target genes were enriched in processes associated with embryo implantation, such as signaling receptor activity, binding and immune response. Nine functional co-expression modules were enriched in various biological processes, such as metabolic pathways, protein transport, cell cycle and VEGF signaling pathway. Additionally, 12 lncRNA-mediated ceRNA networks were constructed. Our results demonstrate that exosomal lncRNAs in uterine flushing fluid exhibit dynamic changes across gestational stages and play an important role in regulating the uterine microenvironment during embryo implantation. These findings provide a foundational basis for screening exosome-derived lncRNAs that influence embryo implantation and contribute to elucidating the mechanistic roles of lncRNAs in exosome-mediated processes during early pregnancy.

embryo implantation↗
PubMed2005

A nuclear surveillance pathway for mRNAs with defective polyadenylation.

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

Catalysis↗
PubMed2003

The roles of intersubunit interactions in exosome stability.

In eukaryotes, at least 10 proteins associate in a 3'-5' exonuclease complex, the exosome, which is involved in the processing of many RNA species. A recent model for the exosome placed six RNase PH-related components in a hexameric ring core structure, with three S1 domain proteins associated with the ring surface. So far, however, this model lacks experimental support. Using a combination of RNA interference, complex affinity purification, and yeast two-hybrid approaches, we show here that the RNase PH homologues are important for maintenance of complex integrity. In contrast, the S1 domain proteins are not required for complex stability, although they are required for exosome function. Our results are partially consistent with the proposed model of the exosome, but indicate a different arrangement of the RNase PH proteins.

Amino Acid Sequence↗
PubMed2004

Regulation of the Neurospora circadian clock by an RNA helicase.

The eukaryotic circadian oscillators consist of autoregulatory negative-feedback loops. FRQ, WC-1, and WC-2 are three known components of the negative-feedback loop of the Neurospora circadian oscillator. FRQ represses its own transcription by interacting with the WC-1/WC-2 complex and inhibiting WC's role in transcriptional activation. Here we show that all FRQ associates with FRH, an essential DEAD box-containing RNA helicase in Neurospora. The budding yeast homolog of FRH, Dob1p/Mtr4p, is a cofactor of exosome, an important regulator of RNA metabolism in eukaryotes. Down-regulation of FRH by inducible expression of a hairpin RNA leads to low levels of FRQ but high levels of frq RNA and the abolishment of circadian rhythmicities. FRH is associated with the WC complex and this interaction is maintained in a frq null strain. Disruption of the FRQ-FRH complex by deleting a domain in FRQ eliminates the FRQ-WC interaction, suggesting that FRH mediates the interaction between FRQ and the WC complex. These data demonstrate that FRH is an essential component in the circadian negative-feedback loop and reveal an unexpected role of an RNA helicase in regulating gene transcription.

Circadian Rhythm↗
PubMed1998

Human dis3p, which binds to either GTP- or GDP-Ran, complements Saccharomyces cerevisiae dis3.

Saccharomyces cerevisiae Dis3p, which interacts with Ran/Gsp1p, complements Schizosaccharomyces pombe dis3-54. Consistent with the functional conservation of Dis3p in S. cerevisiae and S. pombe, the human ORF (accession number: R27667) was found to be highly homologous to yeast Dis3p. Based on its nucleotide sequence, we cloned a full-sized human DIS3 cDNA. The cloned human cDNA partly but significantly restored the temperature-sensitivity of S. cerevisiae dis3. Thus, Dis3p was found to be structurally and functionally conserved from yeast to mammals. Consistent with the report that S. cerevisiae Dis3p is identical to Rrp44p, which comprises the exosome involved in ribosomal RNA processing, S. cerevisiae Dis3p was found to be localized in the nucleolus. Similar to S. cerevisiae Dis3p, human Dis3p enhanced RCC1-stimulated nucleotide release from Ran, in a dose-dependent manner, and bound to GTP- or GDP-Ran.

Amino Acid Sequence↗
PubMed2000

Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA.

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.

Adaptor Proteins, Signal Transducing↗
PubMed2025

Cardiac hypertrophy at the crossroads: Mechanistic insights and emerging multimodal therapeutic strategies.

Cardiovascular diseases (CVDs) remain the leading cause of mortality worldwide, accounting for approximately 17.9 million deaths annually. Among their diverse manifestations, cardiac hypertrophy is a clinically significant condition that predisposes patients to heart failure, arrhythmias, and and sudden cardiac death. Clinically, hypertrophy can be classified into three forms: physiological (adaptive) hypertrophy, which supports cardiac performance and is reversible, pathological hypertrophy most often secondary to hypertension, valvular disease, hemodynamic stress, or sustained neurohumoral activation; and hypertrophic cardiomyopathy (HCM) represents a primary genetic disorder, most often caused by mutations in sarcomeric proteins. These distinct etiologies have important therapeutic implications, as they determine how efficiently pharmacological agents can target underlying mechanisms. Conventional pharmacological treatments are widely used in clinical practice, yet they provide limited reversal of established remodeling. This therapeutic gap has driven the development of innovative modalities such as RNA-based therapeutics, exosome-mediated interventions, stem cell-derived therapies, and genome-editing technologies, which aim to modulate maladaptive signaling and restore myocardial integrity. This review integrates clinical perspectives with mechanistic insights, delineating the drivers of pathological hypertrophy while evaluating both established therapies and emerging strategies that hold promise for precision cardiology and improved patient outcomes.

Humans↗
PubMed2005

Drosophila gene tazman, an orthologue of the yeast exosome component Rrp44p/Dis3, is differentially expressed during development.

The major 3'-5' pathway of RNA degradation in eukaryotic cells involves the exosome, which is a multi-protein complex of exoribonucleases. The exoribonucleases within this complex are highly conserved and are closely related to prokaryotic ribonucleases. We have identified and characterised the expression pattern of Drosophila tazman (taz), a component of the exosome which is closely related to Escherichia coli RNaseR and yeast Rrp44p. The tazman transcripts are differentially expressed during development, with maximum expression levels in 6-8 hr embryos. In situ hybridisation and immunolocalisation experiments show that tazman transcripts and protein are maternally derived, and are expressed ubiquitously throughout the embryo, with high levels in germ band and head structures. Differential expression of TAZ is likely to reflect changes in the activity of the 3'-5' mRNA turnover pathway which could have a major impact of the expression of target RNAs.

Amino Acid Sequence↗
PubMed2001

Quality control of mRNA 3'-end processing is linked to the nuclear exosome.

An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control. Most eukaryotic mRNAs acquire a poly(A) tail during 3'-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm. In the yeast Saccharomyces cerevisiae, a common consequence of defective nuclear export of mRNA is the hyperadenylation of nascent transcripts, which are sequestered at or near their sites of transcription. This implies that polyadenylation and nuclear export are coupled in a step that involves the release of mRNA from transcription site foci. Here we demonstrate that transcripts which fail to acquire a poly(A) tail are also retained at or near transcription sites. Surprisingly, this retention mechanism requires the protein Rrp6p and the nuclear exosome, a large complex of exonucleolytic enzymes. In exosome mutants, hypo- as well as hyperadenylated mRNAs are released and translated. These observations suggest that the exosome contributes to a checkpoint that monitors proper 3'-end formation of mRNA.

Cell Nucleus↗
PubMed2006

A single subunit, Dis3, is essentially responsible for yeast exosome core activity.

The conserved core of the exosome, the major eukaryotic 3' --> 5' exonuclease, contains nine subunits that form a ring similar to the phosphorolytic bacterial PNPase and archaeal exosome, as well as Dis3. Dis3 is homologous to bacterial RNase II, a hydrolytic enzyme. Previous studies have suggested that all subunits are active 3' --> 5' exoRNases. We show here that Dis3 is responsible for exosome core activity. The purified exosome core has a hydrolytic, processive and Mg(2+)-dependent activity with characteristics similar to those of recombinant Dis3. Moreover, a catalytically inactive Dis3 mutant has no exosome core activity in vitro and shows in vivo RNA degradation phenotypes similar to those resulting from exosome depletion. In contrast, mutations in Rrp41, the only subunit carrying a conserved phosphorolytic site, appear phenotypically not different from wild-type yeast. We observed that the yeast exosome ring mediates interactions with protein partners, providing an explanation for its essential function.

Adaptor Proteins, Signal Transducing↗