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.     

Human cell growth requires a functional cytoplasmic exosome, which is involved in various mRNA decay pathways

The human exosome is a 3'-5' exoribonuclease complex that functions both in the nucleus and in the cytoplasm to either degrade or process RNA. Little is known yet about potential differences among core exosome complexes in these different cellular compartments and the roles of the individual subunits in maintaining a stable and functional complex. Glycerol gradient sedimentation analyses indicated that a significant subset of nuclear exosomes is present in much larger complexes (60-80S) than the cytoplasmic exosomes ( approximately 10S). Interestingly, siRNA-mediated knock-down experiments indicated that the cytoplasmic exosome is down-regulated much more efficiently than the nuclear exosome. In addition, we observed that knock-down of hRrp41p or hRrp4p but not PM/Scl-100 or PM/Scl-75 leads to codepletion of other subunits. Nevertheless, PM/Scl-100 and PM/Scl-75 are required to maintain normal levels of three different mRNA reporters: a wild-type beta-globin mRNA, a beta-globin mRNA containing an AU-rich (ARE) instability element, and a beta-globin mRNA bearing a premature termination codon (PTC). The increased levels of ARE- and the PTC-containing mRNAs upon down-regulation of the different exosome subunits, in particular PM/Scl-100, appeared to be due to decreased turnover rates. These results indicate that, although not required for exosome stability, PM/Scl-100 and PM/Scl-75 are involved in mRNA degradation, either as essential subunits of a functional exosome complex or as exosome-independent proteins.     

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.     

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.     

The exosome, a molecular machine for controlled RNA degradation in both nucleus and cytoplasm

One of the most important protein complexes involved in maintaining correct RNA levels in eukaryotic cells is the exosome, a complex consisting almost exclusively of exoribonucleolytic proteins. Since the identification of the exosome complex, seven years ago, much progress has been made in the characterization of its composition, structure and function in a variety of organisms. Although the exosome seems to accumulate in the nucleolus, it has been clearly established that it is also localized in cytoplasm and nucleoplasm. In accordance with its widespread intracellular distribution, the exosome has been implicated in a variety of RNA processing and degradation processes. Nevertheless, many questions still remain unanswered. What are the factors that regulate the activity of the exosome? How and where is the complex assembled? What are the differences in the composition of the nuclear and cytoplasmic exosome? What is the detailed structure of exosome subunits? What are the mechanisms by which the exosome is recruited to substrate RNAs? Here, we summarize the current knowledge on the composition and architecture of this complex, explain its role in both the production and degradation of various types of RNA molecules and discuss the implications of recent research developments that shed some light on the questions above and the mechanisms that are controlling the exosome.     

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.     

Human Mob proteins regulate the NDR1 and NDR2 serine-threonine kinases

Human NDR1 (nuclear Dbf2-related) is a widely expressed nuclear serine-threonine kinase that has been implicated in cell proliferation and/or tumor progression. Here we present molecular characterization of the human NDR2 serine-threonine kinase, which shares approximately 87% sequence identity with NDR1. NDR2 is expressed in most human tissues with the highest expression in the thymus. In contrast to NDR1, NDR2 is excluded from the nucleus and exhibits a punctate cytoplasmic distribution. The differential localization of NDR1 and NDR2 suggests that each kinase may serve distinct functions. Thus, to identify proteins that interact with NDR1 or NDR2, epitope-tagged kinases were immunoprecipitated from Jurkat T-cells. Two uncharacterized proteins that are homologous to the Saccharomyces cerevisiae kinase regulators Mob1 and Mob2 were identified. We demonstrate that NDR1 and NDR2 partially colocalize with human Mob2 in HeLa cells and confirm the NDR-Mob interactions in cell extracts. Interestingly, NDR1 and NDR2 form stable complexes with Mob2, and this association dramatically stimulates NDR1 and NDR2 catalytic activity. In summary, this work identifies a unique class of human kinase-activating subunits that may be functionally analagous to cyclins.     

Neuroglian and DE-cadherin activate independent cytoskeleton assembly pathways in Drosophila S2 cells

The cytoskeletal proteins spectrin and ankyrin colocalize with sites of E-cadherin-mediated cell-cell adhesion in mammalian cells. Here we examined the effects of Drosophila DE-cadherin expression on spectrin and ankyrin in Drosophila S2 tissue culture cells. DE-cadherin caused a dramatic change in the cytoplasmic concentration and distribution of armadillo, the Drosophila homolog of beta catenin. However, DE-cadherin expression had no detectable effect on the quantity or subcellular distribution of ankyrin or spectrin. In reciprocal experiments, recruitment of ankyrin and alphabeta spectrin to the plasma membrane by another cell adhesion molecule, neuroglian, had no effect on the quantity or distribution of armadillo. The results indicate that DE-cadherin-catenin complexes and neuroglian-spectrin/ankyrin complexes form by nonintersecting pathways. Recruitment of spectrin does not appear to be a conserved feature of DE-cadherin function.     

The modular nature of histone deacetylase HDAC4 confers phosphorylation-dependent intracellular trafficking

In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4.14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4.14-3-3 complex.     

Tissue- and developmental stage-specific changes in the subcellular localization of the 26S proteasome in the ovary of Drosophila melanogaster

The intracellular localization of the 26S proteasome in the different ovarian cell types of Drosophila melanogaster was studied by means of immunofluorescence staining and laser scanning microscopy, with the use of antibodies specific for regulatory complex subunits or the catalytic core of the 26S proteasome. During the previtellogenic phase of oogenesis (stages 1-6), strong cytoplasmic staining was observed in the nurse cells and follicular epithelial cells, but the proteasome was not detected in the nuclei of these cell types. The subcellular distribution of the 26S proteasome was completely different in the oocyte. Besides a constant, very faint cytoplasmic staining, there was a gradual nuclear accumulation of proteasomes during the previtellogenic phase of oogenesis. A characteristic subcellular redistribution of the 26S proteasome occurred in the ovarian cells during the vitellogenic phase of oogenesis. There was a gradual decline in the concentration of the 26S proteasome in the nucleus of the oocyte, and in the stage 10 oocyte the proteasome could barely be detected in the nucleus. This was accompanied by a massive nuclear accumulation of proteasomes in the follicular epithelial cells. These results demonstrate that the subcellular distribution of the 26S proteasome in higher eukaryotes is strictly tissue- and developmental stage-specific.     

Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs

Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.     

An exosome-like complex in Sulfolobus solfataricus

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cep1 synthetic lethality 4; an RNA-binding protein and exosome subunit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.     

The C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit

Mitochondrial complex I (CI) is an essential component in energy production through oxidative phosphorylation. Most CI subunits are encoded by nuclear genes, translated in the cytoplasm, and imported into mitochondria. Upon entry, they are embedded into the mitochondrial inner membrane. How these membrane-associated proteins cope with the hydrophilic cytoplasmic environment before import is unknown. In a forward genetic screen to identify genes that cause neurodegeneration, we identified sicily, the Drosophila melanogaster homologue of human C8ORF38, the loss of which causes Leigh syndrome. We show that in the cytoplasm, Sicily preprotein interacts with cytosolic Hsp90 to chaperone the CI subunit, ND42, before mitochondrial import. Loss of Sicily leads to loss of CI proteins and preproteins in both mitochondria and cytoplasm, respectively, and causes a CI deficiency and neurodegeneration. Our data indicate that cytosolic chaperones are required for the subcellular transport of ND42.     

Differential subcellular localization of functionally divergent survivin splice variants

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.