Interdependent Nucleocytoplasmic Trafficking and Interactions of Dis3 with Rrp6, the Core Exosome and Importin‐α3

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‐α3. Surprisingly, dDis3 constructs that lack or mutate the C‐terminal NLS retain importin‐α3 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‐α3 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.     

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.     

The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro

The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.     

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.     

Modulating the RNA Processing and Decay by the Exosome: Altering Rrp44/Dis3 Activity and End-Product

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.     

The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome

Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3′ exonuclease activity. We report that expression only of Rrp44 lacking 3′-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5′-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3′-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo–exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5′-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.     

Dis3‐like 1: a novel exoribonuclease associated with the human exosome

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine‐subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome‐associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity‐purified human exosome complexes identified a novel exosome‐associated exoribonuclease, human Dis3‐like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co‐immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA‐mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)‐tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome‐associated exoribonuclease in the cytoplasm of human cells.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine‐subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R‐like enzyme, which possesses both processive exo‐ and endonuclease activities, whereas the latter is a distributive RNase D‐like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs—hDIS3 and hDIS3L—with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.     

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

Nuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.     

Arabidopsis AtRRP44A Is the Functional Homolog of Rrp44/Dis3, an Exosome Component,Is Essential for Viability and Is Required for RNA Processing and Degradation

The RNA exosome is a multi-subunit complex that is responsible for 3ʹ to 5ʹ degradation and processing of cellular RNA. Rrp44/Dis3 is the catalytic center of the exosome in yeast and humans. However, the role of Rrp44/Dis3 homologs in plants is still unidentified. Here, we show that Arabidopsis AtRRP44A is the functional homolog of Rrp44/Dis3, is essential for plant viability and is required for RNA processing and degradation. We characterized AtRRP44A and AtRRP44B/SOV, two predicted Arabidopsis Rrp44/Dis3 homologs. AtRRP44A could functionally replace S. cerevisiae Rrp44/Dis3, but AtRRP44B/SOV could not. rrp44a knock-down mutants showed typical phenotypes of exosome function deficiency, 5.8S rRNA 3ʹ extension and rRNA maturation by-product over-accumulation, but rrp44b mutants did not. Conversely, AtRRP44B/SOV mutants showed elevated levels of a selected mRNA, on which rrp44a did not have detectable effects. Although T-DNA insertion mutants of AtRRP44B/SOV had no obvious phenotype, those of AtRRP44A showed defects in female gametophyte development and early embryogenesis. These results indicate that AtRRP44A and AtRRP44B/SOV have independent roles for RNA turnover in plants.     

Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p

The RNA exosome processes and degrades RNAs in archaeal and eukaryotic cells. Exosomes from yeast and humans contain two active exoribonuclease components, Rrp6p and Dis3p/Rrp44p. Rrp6p is concentrated in the nucleus and the dependence of its function on the nine-subunit core exosome and Dis3p remains unclear. We found that cells lacking Rrp6p accumulate poly(A)+ rRNA degradation intermediates distinct from those found in cells depleted of Dis3p, or the core exosome component Rrp43p. Depletion of Dis3p in the absence of Rrp6p causes a synergistic increase in the levels of degradation substrates common to the core exosome and Rrp6p, but has no effect on Rrp6p-specific substrates. Rrp6p lacking a portion of its C-terminal domain no longer co-purifies with the core exosome, but continues to carry out RNA 3′-end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated Rrp6-specific rRNA intermediates. However, disruption of Rrp6p–core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products that require the combined activities of Dis3p and Rrp6p. These findings indicate that Rrp6p may carry out some of its critical functions without physical association with the core exosome.     

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.     

Nab3 Facilitates the Function of the TRAMP Complex in RNA Processing via Recruitment of Rrp6 Independent of Nrd1

Non-coding RNAs (ncRNAs) play critical roles in gene regulation. In eukaryotic cells, ncRNAs are processed and/or degraded by the nuclear exosome, a ribonuclease complex containing catalytic subunits Dis3 and Rrp6. The TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex is a critical exosome cofactor in budding yeast that stimulates the exosome to process/degrade ncRNAs and human TRAMP components have recently been identified. Importantly, mutations in exosome and exosome cofactor genes cause neurodegenerative disease. How the TRAMP complex interacts with other exosome cofactors to orchestrate regulation of the exosome is an open question. To identify novel interactions of the TRAMP exosome cofactor, we performed a high copy suppressor screen of a thermosensitive air1/2 TRAMP mutant. Here, we report that the Nab3 RNA-binding protein of the Nrd1-Nab3-Sen1 (NNS) complex is a potent suppressor of TRAMP mutants. Unlike Nab3, Nrd1 and Sen1 do not suppress TRAMP mutants and Nrd1 binding is not required for Nab3-mediated suppression of TRAMP suggesting an independent role for Nab3. Critically, Nab3 decreases ncRNA levels in TRAMP mutants, Nab3-mediated suppression of air1/2 cells requires the nuclear exosome component, Rrp6, and Nab3 directly binds Rrp6. We extend this analysis to identify a human RNA binding protein, RALY, which shares identity with Nab3 and can suppress TRAMP mutants. These results suggest that Nab3 facilitates TRAMP function by recruiting Rrp6 to ncRNAs for processing/degradation independent of Nrd1. The data raise the intriguing possibility that Nab3 and Nrd1 can function independently to recruit Rrp6 to ncRNA targets, providing combinatorial flexibility in RNA processing.     

A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor Activity

Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.