![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 3, 1399-1409, March 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Molecular Biology and Microbiology and Cell Biology Program, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4960
Submitted August 29, 2005;
Revised December 14, 2005;
Accepted January 4, 2005
Monitoring Editor: Marvin P. Wickens
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
; Butler, 2002; Raijmakers et al., 2004). Consistent with its myriad functions, exosome subunits reside in the nucleus and the cytoplasm (Kadowaki et al., 1995; Allmang et al., 1999; Brouwer et al., 2001; Raijmakers et al., 2002b) and exosome complexes can be recovered from extracts representing these subcellular domains (Allmang et al., 1999; Andrulis et al., 2002; Forler et al., 2003). Exosome complexes have been isolated from eukaryotes such as Saccharomyces cerevisiae (Mitchell et al., 1997; Allmang et al., 1999), Drosophila melanogaster (Andrulis et al., 2002; Forler et al., 2003), Arabidopsis thaliana (Chekanova et al., 2000), Homo sapiens (Chen et al., 2001), and Trypanosoma brucei (Estevez et al., 2001; Estevez et al., 2003) as well as from the archaeon Sulfobactus solftaricus (Evguenieva-Hackenburg et al., 2003). The exosome complex is structurally and functionally conserved, as several yeast exosome mutant alleles can be complemented by their cognate human genes (Mitchell et al., 1997; Allmang et al., 1999).
In yeast, the cytoplasmic extract-derived exosome complex ("core") contains 10 subunits in apparent stoichiometry, whereas the "nuclear" complex has one additional subunit, Rrp6 (Ribosomal RNA processing; Allmang et al., 1999). The majority of these subunits are predicted to be 3' to 5' exoribonucleases (Mitchell and Tollervey, 2000). In this regard, purified recombinant Rrp6 and three additional recombinant exosome polypeptides, Rrp4, Rrp44/Dis3, and Rrp41/Ski6, have 3' to 5' exoribonucleolytic activity in vitro (Mitchell et al., 1997; Burkard and Butler, 2000; Chekanova et al., 2000, 2002; Estevez et al., 2001). Several yeast exosome subunits, namely Rrp44/Dis3, Rrp4, Rrp40, and Csl4 contain S1 RNA-binding domains. The reason for grouping so many RNA binding and degradative activities in one protein complex is unclear, although it has been hypothesized that they are important for distinct RNA substrate recognition and regulation (Mitchell and Tollervey, 2000).
Several studies have addressed this hypothesis by examining exosome complex structure. Both high-throughput (Uetz et al., 2000; Ito et al., 2001) and directed (Oliveira et al., 2002; Raijmakers et al., 2002a; Estevez et al., 2003) two-hybrid analyses have revealed specific interactions among and between exosome subunits. These interactions and a three-dimensional (3D) reconstruction of electron microscopic images of the purified yeast exosome complex resulted in a hypothetical structure for exosome core complex (Aloy et al., 2002; Estevez et al., 2003). Recently, the Sulfobactus exosome core was defined to 2.8 Å resolution as a hexameric ring structure consisting of a trimer of Rrp41-Rrp42 heterodimers; the coordination of the exoribonuclease active sites appears to facilitate the recognition and processing of the RNA substrate (Lorentzen and Conti, 2005; Lorentzen et al., 2005). Eukaryotic exosome subunits are thought to assemble in a similar manner in vitro (Lorentzen et al., 2005). In addition, exosome complexes assembled either from the recombinant, purified Archaeoglobus fulgidus (Afu) exosome subunits AfuRrp4, AfuRrp41, and AfuRrp42 or from AfuCsl4, AfuRrp41, and AfuRrp42 are stoichiometric (Buttner et al., 2005).
Despite the fact that exosome subunits can form subcomplexes in vitro, it is generally assumed that all exosome subunits function together in a single complex in vivo. However, whether exosome subunits form subcomplexes in vivo has not been formally addressed. This question requires consideration, because the core and "nuclear" exosome complexes have been exclusively implicated in numerous distinct RNA metabolic pathways. In this regard, although most exosome genes were identified in screens for rRNA processing mutants (Mitchell et al., 1996, 1997), a select few exosome subunits have been shown to localize along transcriptionally active genes (Rrp6, Rrp4, Csl4; Andrulis et al., 2002; Hieronymus et al., 2004). Furthermore, only a few subunits have been implicated in transcription-site retention of aberrant transcripts (Rrp6; Hilleren et al., 2001; Libri et al., 2002), processing or degradation of terminator read-through transcripts (Rrp6 and Rrp41; Torchet et al., 2002), nonstop mRNA decay (Csl4; van Hoof et al., 2002), RNA interference (RNAi)-mediated mRNA turnover (Rrp4, Csl4; Orban and Izaurralde, 2005), NMD-mediated mRNA decay (Rrp4, Rrp41, and Rrp6/PM-Scl100 Lejeune et al., 2003; Mitchell and Tollervey, 2003; Gatfield and Izaurralde, 2004), tRNA surveillance (Rrp44/Dis3; Kadaba et al., 2004), mRNA export (Mtr3 and Rrp6; Kadowaki et al., 1995; Hieronymus et al., 2004), and RNA damage response (Rrp6; Hieronymus et al., 2004). Given that each of these studies focused on specific, rather than all exosome subunits, it is difficult to determine whether functions that are ascribed to the exosome complex are actually mediated by the exosome complex, or are mediated by individual exosome subunits or by subunits in distinct complexes.
To refine our understanding of exosome complex structure and function in vivo, we are studying interactions and localizations of exosome subunits in D. melanogaster S2 cells. Our findings suggest that several distinct exosome subunit subcomplexes exist in vivo.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
) and grown in media supplemented with 300 µg/ml hygromycin B (Invitrogen, Carlsbad, CA).
Cloning, Expression, and Bioinformatic Analysis of Drosophila Exosome Subunits
All plasmids were made using basic molecular cloning techniques. Exosome genes were PCR amplified from full-length cDNAs (Invitrogen) or available laboratory clones using primers with unique restriction enzyme sites and with an in-frame 3' FLAG (DYKDDDK) tag or both 3' FLAG and 6xHis epitope. After digestion with the appropriate enzymes, the tagged exosome genes were ligated into pRmHa3 to obtain tagged exosome genes downstream of the metallothionein (Mtn) promoter. Mtn-exosome constructs were transiently transfected using CELLFectin (Invitrogen) and tested for copper-inducible expression. Maltose-binding protein (MBP) fusions to exosome genes were made similarly, except the genes were cloned into the pMAL-c2 vector (New England Biolabs, Beverly, MA) and the 3' primer lacked the epitope tags. Recombinant MBP-exosome subunit fusions were purified from Escherichia coli using single-step amylose resin affinity purification and maltose elution of MBP fusion proteins, followed by extensive dialysis. Protein domains were identified and sequence alignments were performed using BLAST: http://www.ncbi.nlm.nih.gov/BLAST/. Hypothetical nuclear localization signals were identified by PSORT: http://psort.nibb.ac.jp/.
Antibodies
Polyclonal antibodies were raised against the recombinant polypeptides MBP-dDis3, -dRrp46, and -dRrp40. Recombinant proteins were injected into animals and sera recovered (Pocono Rabbit Farm and Lab, Canadensis, PA). Rabbit anti-dDis3, guinea pig anti-dRrp46, and rabbit anti-dRrp40 bleeds were all compared against preimmune sera from the respective animals to determine specificity. Guinea pig anti-dCsl4, anti-dRrp42, and anti-dRrp4 were affinity-purified against the cognate purified recombinant protein.
Cell Fractionation
Cells were grown to a density of 1 x 107 cells/ml on a 100-mm Petri dish and recovered, centrifuged, washed with wash buffer (10 mM Tris, pH 7.4, 140 mM NaCl), centrifuged, and then lysed, with vigorous pipetting, in the presence of lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 3 mM MgCl2, 0.5 mM EDTA, pH 8.0, 0.5 mM DTT, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail; Invitrogen) on ice for 5 min. After centrifugation, supernatant was removed (
150 µl), the resulting pellet was resuspended in an equivalent volume to the reserved supernatant, and both supernatant and pellet were mixed with 1x SDS loading dye and analyzed by Western blotting.
Immunoprecipitation and Immunoaffinity Purification
For exosome complex immunoaffinity purifications, whole cell extracts were prepared from each of the stable cell lines as described above for the cell fractionation extracts, except cell lysis was for 30 min, and all steps were performed in the presence of 450 NaCl. About 150 µl of soluble extract was obtained from 1 x 108 cells, and 90% of this was placed into an Eppendorf tube with 800 µl wash buffer (lysis buffer but 0.1% nonidet P-40 rather than TX-100), and 20 µl of a 50% slurry of M2 anti-FLAG affinity resin (Sigma, St. Louis, MO) that had been pre-equilibrated in wash buffer. After gentle end-over-end mixing for 1–2 h, beads were spun down, and washed exhaustively, tagged subunits were eluted with FLAG peptide (300 µg/ml), and eluates were resuspended in 1x SDS loading dye for analysis by SDS-PAGE and Western blotting.
|
|
Indirect Immunofluorescence
Stably transfected or untransfected S2 cells were plated in 100-mm Petri dishes and cultured for 1–2 d at early to midlog phase. The stably transfected S2 were induced with the appropriate CuSO4 concentration for 16 h. Cells were collected by centrifugation, washed once in phosphate-buffered saline (PBS), fixed in freshly prepared 3.7% formaldehyde in PBS for 10 min, and then washed once in PBS. All antibodies were diluted in PBS with 0.1% saponin and 1–2% normal donkey serum (PSN; Jackson ImmunoResearch, West Grove, PA). One-fifth of the cells were placed in amber Eppendorf tubes, stained for 1 h in primary antibodies (mouse anti-FLAG [1:500 dilution; Sigma], rabbit anti-dRrp6 [1:500], rabbit anti-dRrp40 [1:100], or affinity-purified guinea pig anti-dCsl4, anti-dRrp42, or anti-dRrp4 [1:5]), washed three times in PBS/0.05% saponin, stained for 45 min in specific secondary antibody (anti-mouse TR, anti-rabbit Cy2, or anti-guinea pig Cy2 at 1:100 dilutions; Jackson ImmunoResearch) and then washed three times with PBS before mounting (in 90% glycerol, 10% 100 mm Tris, pH 8.0, and 0.5% n-propyl gallate). For double immunofluorescence, cells were blocked overnight in PSN and probed with rabbit anti-Drosophila lamin Dm0 (kind gift of Paul A. Fisher, State University of New York, Stony Brook, NY) at 1:2000 and then washed and probed with secondary antibody as described above. DAPI (4'-6-diamidino-2-phenylindole; 1 µg/ml) was added during the second wash of the secondary antibody.
Imaging Analysis
Nondeconvolved images (see Figures 1 and 4) were obtained by mounting cells on 12-well slides (Carlson Scientific, Peotone, IL) and capturing data with a Zeiss Axioplan 2 microscope (Thornwood, NY)/Hamamatsu Digital CCD camera (Bridgewater, NJ). Z-series color images (see Figures 5 and 7) were obtained by mounting cells on cover slips in GelMount (Biomeda) and capturing data with a DeltaVision Restoration microscope system using an Olympus IX70 microscope (Melville, NY) fitted with an automated stage (Applied Precision) and a CCD digital camera. Optical sections (0.2–0.25 µm) were acquired using a 100x (Nikon, PlanApo, NA 0.45 lens) objective lens. The Softworks deconvolution software (Applied Precision, Issaquah, WA) was used to remove out-of-focus light for deconvolved images and to obtain the Pearson coefficient of correlation. For experiment colocalizing lamin and the FLAG-tagged exosome subunits, the nucleus of each cell evaluated was defined as a region of interest through the z-series and then compared for staining intensity of both the FLAG and the lamin stain. All images were arranged using Adobe Photoshop (San Jose, CA).
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
; Burkard and Butler, 2000), and also in the cytoplasm, as has been observed in mammalian cells (Lejeune et al., 2003). In contrast, endogenous dRrp40 and dRrp42 exhibited a diffuse cytoplasmic localization in small cytoplasmic foci and were apparently excluded from the nucleus (Figures 1, B and C). Appropriate preimmune sera showed nonspecific background staining (Supplementary Figure 1). Affinity-purified antibodies to dCsl4 and dRrp4 detected these subunits not only diffuse throughout the cytoplasm but also concentrated in foci that appeared within the cytoplasm and on or around the nucleus (Figure 1, D and E). These data indicate that individual endogenous Drosophila exosome subunits show at least three discrete subcellular localization patterns. A more comprehensive investigation into exosome subunit distribution is therefore of interest.
Expression of Epitope-tagged Exosome Subunits in S2 Cells
Given these distinct immunolocalization patterns of exosome subunits in S2 cells, we next wanted to inquire whether other exosome subunits have similar or different distribution profiles. As a first step toward addressing this issue, we developed a set of stable cell lines expressing individual epitope-tagged exosome subunits (Figure 2A).
Each exosome subunit was epitope tagged with either FLAG (F) or FLAG and 6xHis (FH) and cloned downstream of the metallothionein (Mtn) promoter to allow for copper-inducible expression. After establishment of stable D. melanogaster S2 cell lines, we used Western blot analysis to show that all exosome subunits were up-regulated after copper addition to media (Figure 2B). In several of these cell lines, the tagged exosome subunits were significantly overexpressed relative to the cognate endogenous polypeptides. Tagged exosome subunit overexpression led to the reduction of the cognate endogenous exosome polypeptide (Figures 2C and 3A; as observed in Estevez et al., 2003). Thus, the copper concentration in media was reduced to allow for approximately equal amounts of tagged protein relative to the endogenous protein, as judged by Western blotting (Figure 2C). All subsequent experiments were performed under induction conditions that have been optimized for each epitope-tagged exosome subunit.
|
) indicated that the Drosophila exosome complex is salt-stable. Thus, S2 whole cell extracts were prepared and immunoprecipitations were performed in the presence of 0.45 M NaCl to both extract nucleoplasmic complexes and to assess the stability of the interactions among the Drosophila exosome subunits.
Tagged exosome subunits were purified from whole cell extracts by binding to an anti-FLAG affinity resin. After extensive washing of the resin, complexes were eluted with FLAG peptide. Eluates were analyzed by SDS-PAGE and compared with a loading control (input). Three generally different exosome complex recovery efficiencies are observed. First, dDis3FH, dRrp4FH, dRrp41FH, dRrp40F, and dCsl4FH (Figure 3A, lanes 11, 15, 16, 18, and 19) were most effective at recovering endogenous exosome subunits, with similar amounts of subunits recovered as judged by Western blotting. Second, dRrp6FH and dRrp46F (Figure 3A, lanes 12 and 17) recovered less of each subunit examined. Surprisingly, epitope-tagged dRrp46 recovers endogenous dDis3 better than dRrp6, which is deficient in dDis3 recovery. This would suggest that dDis3 is not stably associated with all exosome subunits, as has been shown for yeast Dis3 (Allmang et al., 1999). Finally, the recovery of endogenous exosome subunits with dMtr3F and dRrp42F was consistently poor (Figure 3A, lanes 13 and 14). Because dRrp42F is expressed rather poorly, this provides an explanation for the low recovery of associated exosome components. Nonetheless, all exosome subunits were coprecipitated specifically, as judged by an inability to recover an unrelated protein, HSF. Moreover, complexes were coprecipitated at levels above background binding to the affinity resin (Figure 3A, lane 20). Importantly, the tagged subunits themselves were recovered with similar efficiencies (Figure 3B), suggesting that these different exosome complex recovery efficiencies are not attributable to an inability to precipitate the tagged polypeptide from whole cell extracts. Thus, tagged exosome subunits incorporate into complexes composed of the endogenous exosome subunits found in whole cell extracts.
To determine the efficiency of endogenous exosome subunit extraction from these cytoplasmic foci and the nucleus, we examined the distribution of endogenous exosome subunits in fractionated cell extracts. Standard cell lysis and fractionation, in buffer containing physiological salt (0.15 M NaCl) and nonionic detergent, elicits a supernatant fraction (S), which includes soluble and cytoplasmic proteins, and a pellet fraction (P), which includes insoluble and nuclear proteins. Under these conditions, endogenous dRrp6 is enriched in the insoluble fraction, and dRrp4, dRrp41, and dCsl4 are predominantly soluble (Supplementary Figure 2A). This solubility profile extends across a range of salt conditions (Supplementary Figure 2B). These data demonstrate that the whole cell extracts prepared for immunoprecipitations in Figure 3 contain the majority of endogenous nuclear and cytoplasmic exosome subunits.
Tagged Exosome Subunits Localize to and Are Enriched in Distinct Subcellular Compartments
Because tagged exosome subunits incorporate into nuclear- and cytoplasmically derived complexes (Figure 3), we surmised that they would localize properly in vivo. To test this idea, we performed indirect immunofluorescence using an antibody to the FLAG epitope to detect each subunit. There was extremely good correlation between the localization of tagged and endogenous exosome polypeptides (cf. Figures 1 and 4). Moreover, consistent with our immunolocalization analysis of endogenous exosome subunits, several distinct tagged subunit localization patterns were observed.
dMtr3F and dRrp40F were observed throughout the cytoplasm and around the nucleus (Figure 4, A and B). An additional set of exosome subunits, dRrp46F, dRrp4FH, dRrp42F, and dCsl4FH were likewise found in the cytoplasm yet specifically enriched in one, two, or more large foci or structures (0.5–3 µm) and/or multiple small foci (0.1–0.4 µm; Figures 4, C–F). These foci vary in size and number depending upon the growth state of the cells and the level of exosome subunit overexpression (Table 1; A. C. Graham and E. D. Andrulis, unpublished observations). Both dDis3FH and dRrp41FH showed restricted localization patterns to either the cytoplasm or the nucleus in individual cells, with dDis3FH predominantly nuclear and dRrp41FH predominantly cytoplasmic (Figure 4, G and H; Table 1). dDis3FH and dRrp41FH also appeared to be enriched around the nucleus. By comparison, dRrp6FH was enriched in the nucleolus (compare immunofluorescence with differential interference contrast panels) and in the nucleus but not in the cytoplasm (Figure 4I). Importantly, indirect immunofluorescence with the anti-FLAG antibody in cells expressing the Mtn vector had background signal that was observed only upon increasing the exposure time (Figure 4J). These images thus reflect the localization of FLAG-tagged exosome subunits and are not due to nonspecific signal from the anti-FLAG antibody.
|
Because these stable cell lines are not clonal and individual subunits showed subtle changes in distribution from cell to cell, we examined 100 cells for the localization of each tagged exosome subunit (Table 1). The most obvious difference uncovered was variation in the number and size of cytoplasmic foci for dRrp42F, dRrp4FH, dRrp46F, and dCsl4FH. In addition, dMtr3F, dRrp40F, and dRrp46F were infrequently found in cytoplasmic regions proximal to the plasma cell membrane. This immunofluorescence analysis shows that individual Drosophila exosome subunits reside in different subcellular locations, with some variation in these localization patterns from cell to cell.
Exosome Subunits Localize to the Nuclear Lamina
Several endogenous and tagged exosome subunits appeared to localize on or around the nuclear rim. Because this localization could correspond to the outer nuclear membrane, inner nuclear membrane, or nuclear lamina, we needed to examine the distribution of exosome subunits relative to an established marker protein. We chose lamin Dm0, a Drosophila B-type lamin that defines the most peripheral part of the nucleoplasm. To obtain higher-resolution images, we used deconvolution microscopy.
Double immunofluorescence detected both lamin Dm0 and FLAG epitopes in the stable S2 cell lines. A portion of dMtr3F localized along parts of the "outside" of the nuclear lamina (Figure 5A). Most strikingly, dDis3FH and dRrp41FH showed appreciable colocalization with lamin that was often restricted to one side of the nucleus, regardless of whether these exosome subunits were nuclear or cytoplasmically localized (Figures 5, G and H; Supplementary Videos 1–3). This is particularly clear in a series of Z-sections taken from a cell expressing dDis3FH (Figure 5J). Although dRrp4FH, dRrp46F, dRrp42F, and dCsl4FH foci often appear proximal to the nuclear rim, modest or negligible costaining with the lamina is observed (Figures 5, C–F; Supplementary Videos 4–6). Unexpectedly, we also observe nuclear foci in cells expressing either dRrp4FH and dRrp42F (Figure 5, D and E; Supplementary Video 5). Lastly, analysis of dRrp6FH localization shows distribution along the outer edges of what is predicted to be the nucleolus and partial overlap with the lamina (Figure 5I).
To determine whether the exosome-lamin colocalization was statistically significant, we calculated the Pearson coefficient of correlation in 10 cell nuclei for each tagged exosome subunit (Figure 6). dRrp6FH, dDis3FH, and dRrp41FH colocalization with lamina scored Pearson coefficients of 0.4 (closest to a score of 1 indicating direct correlation). dMtr3F, dRrp42F, dRrp40F and dRrp4FH had intermediate Pearson coefficients of 0.2, whereas dRrp46F and dCsl4FH scored close to or below zero. These data suggest that several exosome subunits having overlapping localization with lamin in fixed S2 cells.
|
80% of the cells examined (Figure 7A, bottom panels). A similar finding was made with dRrp42F and dCsl4. In this case, however, 75% of the cells exhibited little or no overlap between dRrp42F and dCsl4 (Figure 7B, bottom panel), whereas the remainder of the cells show a high degree of subunit coincidence (Figure 7B, top panel).
Based on these observations, we tested whether Csl4 localization to these foci required Rrp4. Treating S2 cells with dsRNA to dRrp4 reduced endogenous dRrp4 levels to 10% without affecting dCsl4 protein levels (Supplementary Figure 3). Nonetheless, there was no change in dCsl4 subcellular distribution under these conditions (unpublished data). We conclude that dRrp4 is not required for the targeting of dCsl4 to, or for the stability of dCsl4 within, cytoplasmic foci.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). In addition to this proteomic study, another group (Krogan et al., 2004) showed that purified exosome complexes are often missing one or more subunits and contain additional factors that are unique to the precipitated subunit. In this regard, our previous work indicates that the exosome exists in a nuclear complex containing the transcription elongation factor dSpt6 yet lacking the exosome subunit dRrp45 (Andrulis et al., 2002). Finally, consistent with the idea that exosome subunits assemble into distinct complexes, the initial exosome complex comprised 5 subunits in apparent stoichiometry (Mitchell et al., 1997), but the subsequent core complex contained 10 stoichiometric subunits (Allmang et al., 1999).
Recent in vitro studies provide a framework on which to examine our findings. For example, that dRrp41, dRrp42, and dMtr3 are in similar subcellular compartments is consistent with the ability of these subunits to associate in complexes. Indeed, homologues of Rrp42 complex with Rrp41 (Buttner et al., 2005; Lorentzen and Conti, 2005; Lorentzen et al., 2005) and with Mtr3 (Lorentzen et al., 2005). In addition, that dRrp4 and dCsl4 do not always colocalize is consistent with the model that these proteins can assemble either into distinct complexes or into the same complex. Indeed, this is the case with homologues of these subunits in vitro (Buttner et al., 2005). These data indicate an unanticipated diversity of exosome complexes.
Exosome Subunits and Cytoplasmic Foci
That several, but not all, exosome subunits localize to different cytoplasmic foci provides support for cytoplasmic exosome subcomplexes. Two different cytoplasmic structures have been implicated in RNA metabolism: stress granules and processing bodies (P-bodies; also called GW bodies), distinct compartments for mRNA sequestration, storage, or decay (Sheth and Parker, 2003; Cougot et al., 2004; Kedersha et al., 2005). Although the yeast exosome is not known to localize to P-bodies, the entire set of subunits have not been exhaustively examined (Brengues et al., 2005). This is particularly important to explore in light of our findings that only a few of the exosome subunits are enriched in cytoplasmic foci. Interestingly, dRrp4 and dCsl4, both of which localize to cytoplasmic foci in this study, have been shown to be required for RNAi-mediated mRNA decay (Orban and Izaurralde, 2005). Notably, the RNA-induced silencing complex machinery, required for degrading RNAi-targeted mRNAs, localizes to P-bodies in mammalian cells (Liu et al., 2005). Thus, we favor the idea that some of these exosome subunit cytoplasmic foci correspond to P-bodies. However, this explanation is insufficient to explain the observation that dRrp4FH and dRrp42F are largely in cytoplasmic compartments distinct those containing dCsl4. Thus, the composition and functions of these cytoplasmic structures awaits identification.
Are Exosome Subunits Positioned on the Nuclear Lamina for mRNA Surveillance?
The localization of several exosome subunits to the nuclear lamina is likewise interesting. It is possible that nucleoperipheral domain represents a storage or nucleocytoplasmic transit site for exosome subunits. An alternative possibility is that exosome subunits at the lamina are critical for surveillance during mRNA export (Kadowaki et al., 1995; Hilleren et al., 2001; Jensen et al., 2001; Zenklusen et al., 2002; Thomsen et al., 2003; Galy et al., 2004; Hieronymus et al., 2004). Interestingly, two of the lamina-localizing exosome subunits were identified as yeast mutants (mtr3 and rrp6) defective for mRNA export (Kadowaki et al., 1995; Hieronymus et al., 2004). The asymmetric laminar localization of dRrp41 and dDis3 is reminiscent of the yeast myosinlike proteins (Mlp), factors critical for the nuclear retention of aberrant mRNAs (Galy et al., 2004; Vinciguerra et al., 2005). Because the yeast exosome proteins Rrp41/Ski6 and Dis3/Rrp44 are required for turnover of these aberrant mRNAs in the nucleus (Bousquet-Antonelli et al., 2000), it is appealing to speculate that these asymmetric, lamina-directed exosome subunits may function in a similar mRNA export and surveillance pathway in Drosophila cells.
Differential Localization of Exosome Subunits
Both individual endogenous and epitope-tagged exosome subunits localize differentially in Drosophila S2 cells. Although several exosome subunits in yeast and mammalian cells fractionate and localize similarly to their Drosophila homologues (Kadowaki et al., 1995; Allmang et al., 1999; Brouwer et al., 2001; Raijmakers et al., 2002b), others do not (Brouwer et al., 2001; Raijmakers et al., 2002b). Nevertheless, we find it unlikely that the epitope-tagged Drosophila exosome subunits mislocalize, as they localize similarly to the cognate endogenous protein and incorporate into exosome complexes.
We find exosome subunits specifically enriched at the nucleolus, nucleus, nuclear foci, nuclear rim, cytoplasm, cytoplasmic foci, and plasma cell membrane. Why so many distinct locales? There are several possibilities. First, perhaps exosome subunits are dynamic, and our localization analysis in fixed cells provides a limited perspective. In one particular example, the nuclear- or cytoplasmic-restricted localization patterns of dDis3FH and dRrp41FH suggest that these two proteins may shuttle between the nucleus and cytoplasm. However, treating cells with the drug leptomycin B does not change dDis3FH and dRrp41FH distribution (unpublished data), indicating that these proteins do not use a Crm1-dependent export pathway. Interestingly, Schizosaccharomyces pombe and human Dis3 interact directly with Ran (Noguchi et al., 1996; Shiomi et al., 1998), a protein essential for nucleocytoplasmic transport of protein and ribonucleoprotein cargoes (Macara, 2001). Second, exosome subunits may localize to different subcellular domains depending on changes in the cell cycle or growth conditions. Third, individual exosome subunits, singly or in combination with other subunits, may occupy specific subcellular domains crucial for specialized roles in mRNA processing, turnover, transport, or surveillance or in the metabolism of other RNA species. Finally, exosome subunits may reside in complexes distinct from the exosome complex, where they perform functions that are unrelated to RNA regulation. In this regard, Dis3 has a mitotic role in fission yeast (Kinoshita et al., 1991) and Rrp6 is implicated in DNA damage surveillance and recombination in budding yeast (Hieronymus et al., 2004; Luna et al., 2005).
To date, most studies on exosome function have utilized either mutants or RNAi-depletions of one or two subunits to suggest a role for the core or nuclear exosome complex in a particular pathway. Our results provide compelling evidence for the existence of several distinct, subcellularly compartmentalized exosome subcomplexes. In so doing, this study promotes the idea that exosome subunits, singly or in exosome subunit subcomplexes, function in several of the RNA metabolic pathways that have been ascribed to the exosome complex.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Erik D. Andrulis (erik.andrulis{at}case.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
5' exonucleases. Genes Dev. 13, , 2148–2158.Aloy, P., Ciccarelli, F. D., Leutwein, C., Gavin, A. C., Superti-Furga, G., Bork, P., Bottcher, B., and Russell, R. B. ((2002). ). A complex prediction: three-dimensional model of the yeast exosome. EMBO Rep. 3, , 628–635.[CrossRef][Medline]
Andrulis, E. D., Werner, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P., and Lis, J. T. ((2002). ). The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature 420, , 837–841.[CrossRef][Medline]
Bousquet-Antonelli, C., Presutti, C., and Tollervey, D. ((2000). ). Identification of a regulated pathway for nuclear pre-mRNA turnover. Cell 102, , 765–775.[CrossRef][Medline]
Brengues, M., Teixeira, D., and Parker, R. ((2005). ). Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, , 486–489.
Brouwer, R., Allmang, C., Raijmakers, R., van Aarssen, Y., Egberts, W. V., Petfalski, E., van Venrooij, W. J., Tollervey, D., and Pruijn, G. J. ((2001). ). Three novel components of the human exosome. J. Biol. Chem. 276, , 6177–6184.
Burkard, K. T., and Butler, J. S. ((2000). ). A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p. Mol. Cell. Biol. 20, , 604–616.
Butler, J. S. ((2002). ). The yin and yang of the exosome. Trends Cell Biol. 12, , 90–96.[CrossRef][Medline]
Buttner, K., Wenig, K., and Hopfner, K. P. ((2005). ). Structural framework for the mechanism of archaeal exosomes in RNA processing. Mol. Cell 20, , 461–471.[CrossRef][Medline]
Bycroft, M., Hubbard, T. J., Proctor, M., Freund, S. M., and Murzin, A. G. ((1997). ). The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. Cell 88, , 235–242.[CrossRef][Medline]
Chekanova, J. A., Dutko, J. A., Mian, I. S., and Belostotsky, D. A. ((2002). ). Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3'5' exonuclease containing S1 and KH RNA-binding domains. Nucleic Acids Res. 30, , 695–700.
Chekanova, J. A., Shaw, R. J., Wills, M. A., and Belostotsky, D. A. ((2000). ). Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells. J. Biol. Chem. 275, , 33158–33166.
Chen, C. Y., Gherzi, R., Ong, S. E., Chan, E. L., Raijmakers, R., Pruijn, G. J., Stoecklin, G., Moroni, C., Mann, M., and Karin, M. ((2001). ). AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell 107, , 451–464.[CrossRef][Medline]
Clissold, P. M., and Ponting, C. P. ((2000). ). PIN domains in nonsense-mediated mRNA decay and RNAi. Curr. Biol. 10, , R888–R890.[CrossRef][Medline]
Cougat, N., Babajko, S., and Seraphin, B. ((2004). ). Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165, , 31–40.
Estevez, A. M., Kempf, T., and Clayton, C. ((2001). ). The exosome of Trypanosoma brucei. EMBO J. 20, , 3831–3839.[CrossRef][Medline]
Estevez, A. M., Lehner, B., Sanderson, C. M., Ruppert, T., and Clayton, C. ((2003). ). The roles of intersubunit interactions in exosome stability. J. Biol. Chem. 278, , 34943–34951.
Evguenieva-Hackenburg, E., Walter, P., Hochleitner, E., Lottspeich, F., and Klug, G. ((2003). ). An exosome-like complex in Sulfolobus solfataricus. EMBO Rep. 4, , 889–893.[CrossRef][Medline]
Forler, D., Kocher, T., Rode, M., Gentzel, M., Izaurralde, E., and Wilm, M. ((2003). ). An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat. Biotechnol. 21, , 89–92.[CrossRef][Medline]
Galy, V., Gadal, O., Fromont-Racine, M., Romano, A., Jacquier, A., and Nehrbass, U. ((2004). ). Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1. Cell 116, , 63–73.[CrossRef][Medline]
Gatfield, D., and Izaurralde, E. ((2004). ). Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, , 575–578.[CrossRef][Medline]
Gavin, A. C. et al. ((2002). ). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, , 141–147.[CrossRef][Medline]
Hieronymus, H., Yu, M. C., and Silver, P. A. ((2004). ). Genome-wide mRNA surveillance is coupled to mRNA export. Genes Dev. 18, , 2652–2662.
Hilleren, P., McCarthy, T., Rosbash, M., Parker, R., and Jensen, T. H. ((2001). ). Quality control of mRNA 3'-end processing is linked to the nuclear exosome. Nature 413, , 538–542.[CrossRef][Medline]
Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. ((2001). ). A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98, , 4569–4574.
Jensen, T. H., Boulay, J., Rosbash, M., and Libri, D. ((2001). ). The DECD box putative ATPase Sub2p is an early mRNA export factor. Curr. Biol. 11, , 1711–1715.[CrossRef][Medline]
Kadaba, S., Krueger, A., Trice, T., Krecic, A. M., Hinnebusch, A. G., and Anderson, J. ((2004). ). Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae. Genes Dev. 18, , 1227–1240.
Kadowaki, T., Schneiter, R., Hitomi, M., and Tartakoff, A. M. ((1995). ). Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae. Mol. Biol. Cell 6, , 1103–1110.[Abstract]
Kedersha et al. ((2005). ). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169, , 871–884.
Kinoshita, N., Goebl, M., and Yanagida, M. ((1991). ). The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control. Mol. Cell. Biol. 11, , 5839–5847.
Krogan, N. J. et al. ((2004). ). High-definition macromolecular composition of yeast RNA-processing complexes. Mol. Cell 13, , 225–239.[CrossRef][Medline]
Lejeune, F., Li, X., and Maquat, L. E. ((2003). ). Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol. Cell 12, , 675–687.[CrossRef][Medline]
Libri, D., Dower, K., Boulay, J., Thomsen, R., Rosbash, M., and Jensen, T. H. ((2002). ). Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation. Mol. Cell. Biol. 22, , 8254–8266.
Liu, J., Valencia-Sanchez, M. A., Hannon, G. J., and Parker, R. ((2005). ). MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7, , 719–723.[CrossRef][Medline]
Lorentzen, E., and Conti, E. ((2005). ). Structural basis of 3' end RNA recognition and exoribonucleolytic cleavage by an exosome RNase PH core. Mol. Cell 20, , 473–481.[CrossRef][Medline]
Lorentzen, E., Walter, P., Fribourg, S., Evguenieva-Hackenberg, E., Klug, G., and Conti, E. ((2005). ). The archaeal exosome core is a hexameric ring structure with three catalytic subunits. Nat. Struct. Mol. Biol. 12, , 575–581.[CrossRef][Medline]
Luna, R., Jimeno, S., Marin, M., Huertas, P., Garcia-Rubio, M., and Aguilera, A. ((2005). ). Interdependence between transcription and mRNP processing and export, and its impact on genetic stability. Mol. Cell 18, , 711–722.[CrossRef][Medline]
Macara, I. G. ((2001). ). Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, , 570–594, table of contents.
Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M., and Tollervey, D. ((1997). ). The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'5' exoribonucleases. Cell 91, , 457–466.[CrossRef][Medline]
Mitchell, P., Petfalski, E., and Tollervey, D. ((1996). ). The 3' end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism. Genes Dev. 10, , 502–513.
Mitchell, P., and Tollervey, D. ((2000). ). Musing on the structural organization of the exosome complex. Nat. Struct. Biol. 7, , 843–846.[CrossRef][Medline]
Mitchell, P., and Tollervey, D. ((2003). ). An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'5' degradation. Mol. Cell 11, , 1405–1413.[CrossRef][Medline]
Noguchi, E. et al. ((1996). ). Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1. EMBO J. 15, , 5595–5605.[Medline]
Oliveira, C. C., Gonzales, F. A., and Zanchin, N. I. ((2002). ). Temperature-sensitive mutants of the exosome subunit Rrp43p show a deficiency in mRNA degradation and no longer interact with the exosome. Nucleic Acids Res. 30, , 4186–4198.
Orban, T. I., and Izaurralde, E. ((2005). ). Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome. RNA 11, , 459–469.
Phillips, S., and Butler, J. S. ((2003). ). Contribution of domain structure to the RNA 3' end processing and degradation functions of the nuclear exosome subunit Rrp6p. RNA 9, , 1098–1107.
Raijmakers, R., Egberts, W. V., van Venrooij, W. J., and Pruijn, G. J. ((2002a). ). Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring. J. Mol. Biol. 323, , 653–663.[CrossRef][Medline]
Raijmakers, R., Noordman, Y. E., van Venrooij, W. J., and Pruijn, G. J. ((2002b). ). Protein-protein interactions of hCsl4p with other human exosome subunits. J. Mol. Biol. 315, , 809–818.[CrossRef][Medline]
Raijmakers, R., Schilders, G., and Pruijn, G. J. ((2004). ). The exosome, a molecular machine for controlled RNA degradation in both nucleus and cytoplasm. Eur. J. Cell Biol. 83, , 175–183.[CrossRef][Medline]
Sheth, U., and Parker, R. ((2003). ). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, , 805–808.
Shiomi, T., Fukushima, K., Suzuki, N., Nakashima, N., Noguchi, E., and Nishimoto, T. ((1998). ). Human dis3p, which binds to either GTP- or GDP-Ran, complements Saccharomyces cerevisiae dis3. J. Biochem. (Tokyo) 123, , 883–890.
Thomsen, R., Libri, D., Boulay, J., Rosbash, M., and Jensen, T. H. ((2003). ). Localization of nuclear retained mRNAs in Saccharomyces cerevisiae. RNA 9, , 1049–1057.
Torchet, C., Bousquet-Antonelli, C., Milligan, L., Thompson, E., Kufel, J., and Tollervey, D. ((2002). ). Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs. Mol. Cell 9, , 1285–1296.[CrossRef][Medline]
Uetz, P. et al. ((2000). ). A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, , 623–627.[CrossRef][Medline]
van Hoof, A., Frischmeyer, P. A., Dietz, H. C., and Parker, R. ((2002). ). Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science 295, , 2262–2264.
Vinciguerra, P., Iglesias, N., Camblong, J., Zenklusen, D., and Stutz, F. ((2005). ). Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export. EMBO J. 24, , 813–823.[CrossRef][Medline]
Zenklusen, D., Vinciguerra, P., Wyss, J. C., and Stutz, F. ((2002). ). Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p. Mol. Cell. Biol. 22, , 8241–8253.
This article has been cited by other articles:
|
|
 |
|
  Y.-S. Kuan, P. Brewer-Jensen, W.-L. Bai, C. Hunter, C. B. Wilson, S. Bass, J. Abernethy, J. S. Wing, and L. L. Searles Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs Mol. Cell. Biol., October 15, 2009; 29(20): 5590 - 5603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Graham, D. L. Kiss, and E. D. Andrulis Core Exosome-independent Roles for Rrp6 in Cell Cycle Progression Mol. Biol. Cell, April 15, 2009; 20(8): 2242 - 2253. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Callahan and J. S. Butler Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p Nucleic Acids Res., December 1, 2008; 36(21): 6645 - 6655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lange, S. Holec, V. Cognat, L. Pieuchot, M. Le Ret, J. Canaday, and D. Gagliardi Degradation of a Polyadenylated rRNA Maturation By-Product Involves One of the Three RRP6-Like Proteins in Arabidopsis thaliana Mol. Cell. Biol., May 1, 2008; 28(9): 3038 - 3044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-J. Lin, A. Duffy, and C.-Y. Chen Localization of AU-rich Element-containing mRNA in Cytoplasmic Granules Containing Exosome Subunits J. Biol. Chem., July 6, 2007; 282(27): 19958 - 19968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Murray and D. R. Schoenberg A+U-Rich Instability Elements Differentially Activate 5'-3' and 3'-5' mRNA Decay Mol. Cell. Biol., April 15, 2007; 27(8): 2791 - 2799. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Stoecklin and P. Anderson In a tight spot: ARE-mRNAs at processing bodies Genes & Dev., March 15, 2007; 21(6): 627 - 631. [Full Text] [PDF]
|
|
|
|
|
|
T. M. Franks and J. Lykke-Andersen TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements Genes & Dev., March 15, 2007; 21(6): 719 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. Hooker, P. Lam, H. Zheng, and L. Kunst A Core Subunit of the RNA-Processing/Degrading Exosome Specifically Influences Cuticular Wax Biosynthesis in Arabidopsis PLANT CELL, March 1, 2007; 19(3): 904 - 913. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Anderson, D. Mukherjee, K. Muthukumaraswamy, K. C.M. Moraes, C. J. Wilusz, and J. Wilusz Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome RNA, October 1, 2006; 12(10): 1810 - 1816. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-Exo, anti-exosome subunit antibody. DAPI, 4'-6-diamidino-2-phenylindole. DIC, differential interference contrast.
