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1 School of Biological Sciences, University of Missouri–Kansas City, Kansas City, Missouri 64110, USA
2 Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Keywords: mRNA export; mRNP; Saccharomyces cerevisiae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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; Fasken and Corbett 2005). Indeed, Saccharomyces cerevisiae strains bearing temperature-sensitive mutations in genes encoding nuclear processing factors manifest a pronounced nuclear mRNA retention. These genes include poly(A) polymerase PAP1 (Hilleren et al. 2001), the mRNA export receptor MEX67 (Jensen et al. 2001b), and 3'-end processing factors RNA14 and RNA15 (Libri et al. 2002), as well as the components of the nuclear THO/TREX complex SUB2, HPR1, and MFT1 (Jensen et al. 2001a; Libri et al. 2002). Upon a shift to nonpermissive conditions, these strains accumulate mRNA in discrete, transcription-site-proximal foci (hereafter referred to as "dots") that can be visualized by fluorescent in situ hybridization (FISH) with gene-specific probes.
Nuclear mRNA dots can also be observed in wild-type cells under physiological conditions. For example, robust, intense dots arise from a GAL-driven GFP-encoding reporter construct, which bypasses normal 3'-end processing because it terminates in a hammerhead ribozyme (GAL-GFP-RZ). Dots also arise from a reporter with a wild-type GAL 3'-UTR (containing GAL1 3'-end formation signals; GAL-GFP-pA) and even from the endogenous GAL1 gene (Dower et al. 2004; Abruzzi et al. 2006). Therefore, dot formation likely reflects a regular feature of gene expression, which is quantitatively increased when nuclear mRNA processing is suboptimal or perturbed. Importantly, both GAL-GFP-RZ and GAL-GFP-pA reporters give rise to dots containing RNA that is largely post-transcriptional (i.e., non-nascent). This is because the dots are spatially distinct from their transcription sites and because they persist long after the transcriptional shutoff (Abruzzi et al. 2006). Moreover, dots remain adjacent to their transcription sites during the shutoff.
Intriguingly, dot formation correlates with the tendency of active genes to associate with the nuclear periphery (Abruzzi et al. 2006), which suggests mechanistic links between these two processes. Several mechanisms have been proposed to contribute to the recruitment, capture, and/or subperipheral retention of genes at the nuclear envelope. These include direct interactions between transcriptional activators and nucleoporins (Menon et al. 2005; Schmid et al. 2006), the act of transcription itself (Cabal et al. 2006; Taddei et al. 2006), transcription-associated chromatin remodeling (Brickner et al. 2007), as well as unspecified mRNA- and/or 3'-UTR-dependent interactions (Casolari et al. 2005; Taddei et al. 2006). Our own experiments have also highlighted the importance of 3'-end formation signals on gene-periphery associations. Moreover, these have a strong and parallel influence on dot formation (Abruzzi et al. 2006).
In this study, we conducted a FISH-based screen for dot tethering factors. Our strategy was based on the assumption that perturbation of the tether(s) between the dot and its gene would probably impact dot morphology. Indeed, we identified Sus1, Thp1, and Sac3 as factors affecting dot morphology as well as the persistent tethering of dots to their cognate genes after transcriptional shutoff. This strongly implicates the Sac3-Thp1-Sus1-Cdc31 complex in post-transcriptional dot-gene tethering. Remarkably, the association of the endogenous GAL1 locus to the nuclear periphery previously has been shown to be inhibited by the absence of this same complex (Cabal et al. 2006; Drubin et al. 2006). However, its dual association with the transcription coactivator SAGA as well as with NPCs has precluded discriminating between a transcriptional and a post-transcriptional role of the complex. The findings reported here favor the latter, because the retention of previously activated (but transcriptionally silent) GAL-promoter-driven reporter genes at the nuclear periphery similarly requires the activity of these three proteins. The parallel findings on these two tethering phenomena underscore the mechanistic coupling between the mRNA-to-gene and gene-nuclear periphery interactions and emphasize the contribution of post-transcriptional events to both processes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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), which was compiled via an extensive manual survey of genes implicated in mRNA biogenesis, processing, and export (Table 1).
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80% of cells. Importantly, these effects were not dependent on the specific construct or on its integration site, because substituting the conditional GAL1 promoter for the TDH3 promoter as well as placing the reporter at a different genomic location led to an identical dot enlargement and fragmentation in an sus1
background (Fig. 1C). Moreover, simultaneous visualization of the GAL-GFP-RZ locus using TetR-GFP bound to a tandem array of 448 Tet operators integrated <5 kb from the reporter construct (TetR-GFP/TetO448 system) showed that one major dot was always adjacent to the reporter gene (Fig. 1C). (In these and subsequent experiments, the nuclear periphery was simultaneously visualized using Nup49-GFP, which by itself has no effect on the dot morphology.) This suggests a precursor–product relationship between the primary, locus-proximal mRNA dot and secondary dots that break off and diffuse away. Introducing a wild-type (WT) copy of the SUS1 gene into the sus1 strain fully rescued this phenotype (Fig. 1D) as well as the other associated phenotypes described below.
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). Sus1 also copurifies with the transcriptional coactivator SAGA and is believed to mediate transcription-coupled mRNA export (Rodriguez-Navarro et al. 2004). Indeed, loss of Sus1 compromises transcription of a subset of SAGA-regulated genes and also leads to increased accumulation of poly(A)+ RNA in the nucleus (Fischer et al. 2002, 2004; Rodriguez-Navarro et al. 2004). Because the TDH3 promoter used to drive the GFP-RZ reporter is SAGA-regulated (Basehoar et al. 2004), we determined whether the sus1 effect on dot morphology is principally due to the impaired function of SAGA or of the Sac3-Thp1-Sus1-Cdc31 complex. To this end, we tested by FISH four additional deletion strains missing components of these complexes.
We found that deletions of the SAGA genes GCN5 or SPT20 had no effect on TDH-GFP-RZ dot morphology (Fig. 1E,F; data not shown). The spt20 mutation causes severe disruption of SAGA complex integrity and function (Grant et al. 1997; Sterner et al. 1999) and prevents Sus1 from associating with the promoter of GAL1 (Kohler et al. 2006). Moreover, the effect of gcn5 on transcription is quantitatively identical to that of sus1 (Fig. 3C,D below; Dudley et al. 1999). We therefore conclude that the SAGA complex does not contribute substantially to mRNP dot morphology.
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cells (Fig. 1G,H), that is, an exaggerated and/or fragmented dot. We conclude that compromising the function of the Sac3-Thp1-Sus1-Cdc31 complex perturbs the transcription-site-proximal GFP-RZ mRNP pool, which is visualized by FISH as an exaggerated and/or fragmented dot. Importantly, this effect is specific, since other mutants that affect poly(A)+ mRNA export to a comparable or greater extent (e.g., lrp1) (Hieronymus et al. 2004; data not shown) had no effect on dot morphology.
The Sac3-Thp1-Sus1-Cdc31 complex impacts the dot-gene tether
To further explore the role of the Sac3-Thp1-Sus1-Cdc31 complex in the tethering of the mRNA dot to its cognate gene during transcription as well as after the transcriptional shutoff, we simultaneously monitored the locations of the dots with FISH and the reporter locus with the TetR-GFP/TetO448 system as described above. As previously reported, the dot as well as its tether to the reporter gene persist for at least 60 min after transcriptional shutoff in WT cells (Abruzzi et al. 2006). In the sus1, sac3, and thp1 mutant strains, however, we observed that the GAL-GFP-RZ dots progressively detach from their loci of origin after transcriptional shutoff (Fig. 2). To extend this observation, we also tested the GAL-GFP-pA reporter construct integrated at the same genomic location. GAL-GFP-pA construct possesses a normal GAL1 3'-UTR and polyadenylation signal (while its GFP chromophore has been inactivated to enable visualization of TetR-GFP/TetO448), and the GAL-GFP-pA dot appears normal during active transcription (Fig. 1I). However, it similarly detaches from its gene in the three deletion strains after transcriptional shutoff (Fig. 2). These data indicate that the Sac3-Thp1-Sus1-Cdc31 complex contributes to retention of a post-transcriptional dot near its gene of origin. Moreover, the lack of an effect before transcriptional shutoff suggests that there are additional, Sac3-, Thp1-, and Sus1-independent mechanisms contributing to the dot-gene tether during active transcription.
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). These differences presumably reflect the different RNP compositions of the two transcripts (see Discussion).
Motivated by these observations, we examined the relationship of the reporter loci to the nuclear periphery during active transcription in the sus1, sac3, and thp1 strains as well as peripheral retention after transcriptional shutoff. In contrast to WT cells (Abruzzi et al. 2006), the GAL-GFP-pA gene locus completely failed to localize to the nuclear rim under activating conditions in the deletion strains (galactose) (Fig. 3A). This recapitulates perfectly the behavior of the endogenous GAL1 locus in these strains (Cabal et al. 2006; Drubin et al. 2006). In contrast, the GAL-GFP-RZ gene still localized to the nuclear periphery in the deletion strains. However, its association was rapidly lost upon transcriptional shutoff, with kinetics that paralleled that of RNAP II runoff (Fig. 3B). This contrasts with WT cells, in which the GAL-GFP-RZ locus dissociates from the nuclear rim only very slowly after glucose addition (>60 min) (Abruzzi et al. 2006). The results suggest the existence of additional transcription-dependent tether(s) that maintain an actively transcribing GAL-GFP-RZ locus at the nuclear periphery. They also suggest that the post-transcriptional retention of the GAL-GFP-RZ locus at the nuclear periphery requires the Sac3-Thp1-Sus1-Cdc31 complex.
Because Sus1 directly participates in transcription from the GAL1 promoter (Rodriguez-Navarro et al. 2004; Kohler et al. 2006), we addressed the possibility that the differences in intranuclear positioning between the active GAL-GFP-pA and GAL-GFP-RZ genes in the sus1 mutant is a consequence of differential transcriptional effects on these two genes. To this end, we compared the RNAP II occupancy on these two reporter constructs, using chromatin immunoprecipitation (ChIP). The magnitude of reduction in RNAP II occupancy of GAL-GFP-RZ and GAL-GFP-pA in sus1 relative to WT cells was identical (Fig. 3C), and it was also identical to that of the endogenous GAL1 gene in sus1 cells (data not shown). Moreover, the decrease was quantitatively mirrored by the steady-state mRNA level differences of both reporter genes between the two strains (Fig. 3D).
We also found that a loss of Gcn5, which associates with the GAL1 promoter (Dhasarathy and Kladde 2005) and impacts its transcriptional activity comparably to sus1 (approximately threefold) (Fig. 3C,D; Dudley et al. 1999), had no effect on peripheral retention of the GAL-GFP-pA gene (Fig. 3A). Moreover, it had no more than a modest effect on the dissociation kinetics of the GAL-GFP-RZ locus from the nuclear rim (Fig. 3B). We conclude that the differential impact of an Sus1 deletion on the morphology of GAL-GFP-RZ and GAL-GFP-pA mRNA dots as well as on the nuclear rim association of these genes is not due to its SAGA-related functions but to the Sac3-Thp1-Sus1-Cdc31 complex. We also speculate that post-transcriptional mRNP may more generally facilitate gene repositioning to the nuclear periphery.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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; Gallardo et al. 2003; Lei et al. 2003; Rodriguez-Navarro et al. 2004; Kohler et al. 2006).
Characterization of the sus1, thp1, and sac3 strains indicates that fragmentation of the GAL-GFP-RZ mRNP dot is intimately related to weakening of the gene-dot tethering. This might indicate that dot integrity is dependent on contacts between dot mRNP, Sac3-Thp1-Sus1-Cdc31 complex, and chromatin. When these are diminished (e.g., in sus1), dot fragmentation as well as separation of the dot and the gene occur (Figs. 1, 4). Notably, despite their fragmentation in the mutant backgrounds, the GAL-GFP-RZ dots do not completely disperse and disappear, indicating that there must be Sus1-, Sac3-, and Thp1-independent associations between the individual mRNP particles within the dot that contribute to its integrity.
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, sac3, and thp1 mutations strongly impact the association of the GAL-GFP-pA gene with the nuclear rim. Yet their effects on this reporter differ in two respects from their effects on GAL-GFP-RZ: the GAL-GFP-pA dots are neither enlarged nor fragmented in the mutants, and the association of the GAL-GFP-pA gene with the nuclear periphery is abolished completely, i.e., even during active transcription. As only the GAL-GFP-RZ gene remains associated with the nuclear rim during active transcription in the mutant strains, there must be additional transcription-dependent contacts that are stronger or more numerous between the GAL-GFP-RZ gene and the nuclear periphery.
Although we cannot rule out that such differential contacts may be mediated by chromatin per se, there are no known chromatin effects associated with replacing the GAL1 3'-UTR with the ribozyme. An alternative (or additional) possibility would be due to a difference between GAL-GFP-pA and GAL-GFP-RZ mRNP structure or composition, which can be qualitative, quantitative, or both. Indeed, we reported previously that the GAL-GFP-RZ gene shows an altered cotranscriptional recruitment profile of Yra1 (Abruzzi et al. 2006). Moreover, a quantitative difference is indicated by the consistently larger GAL-GFP-RZ dots than the GAL-GFP-pA dots (data not shown; D. Zenklusen and R. Singer, pers. comm.). Larger dots would provide more contact area between RZ mRNP and the nuclear periphery than between pA RNP and the periphery, which might contribute to RZ gene retention at the nuclear rim during active transcription even in a Sus1-deleted strain (Fig. 4).
This view implies that there are multiple contacts between a tethered gene and the NPC. Indeed, it has been shown that transcriptional activator binding (Schmid et al. 2006), transcription-associated chromatin remodeling (Brickner et al. 2007), and perhaps the act of transcription itself (Casolari et al. 2005; Cabal et al. 2006; Drubin et al. 2006) all contribute to the recruitment of the activated GAL genes to the nuclear periphery. Moreover, diminished perinuclear positioning during active transcription was observed for endogenous GAL genes in sus1 and sac3 cells (e.g., Cabal et al. 2006; Drubin et al. 2006). Our findings extend these conclusions by suggesting that this effect is mediated by the post-transcriptional mRNP and becomes relevant only after initial contact of the activated gene with the nuclear rim (modeled in Fig. 4). Whereas the initial encounter of the locus with the nuclear periphery is transcriptional activator-dependent, independent of Sus1 (Schmid et al. 2006), and precedes the onset of transcription (Brickner et al. 2007), stable retention becomes independent of active transcription and is facilitated by the Sac3-Thp1-Sus1-Cdc31 complex; it is more generally aided by intrinsic properties of post-transcriptional mRNP. We suggest that multiple tethering mechanisms serve to strengthen the association between the active gene and NPC and hence facilitate rapid mRNA export, as originally proposed by Blobel in the gene gating hypothesis (Blobel 1985).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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). Reporter strains designed for mating with deletion array strains were in a Y7092 background (MAT
can1::STE2pr-Sphis5 his31 leu20 ura30 met150 lyp1 trp1::GAL1-IpgB1-URA3) (Alto et al. 2006; gift from Charlie Boone).
Yeast strains other than the ones used in the FISH-based screen for altered dot morphology as well as their derivation are described in detail in Table 2. To delete the SUS1, SAC3, and THP1, the kanamycin (KanMX) cassette plus respective flanking regions were PCR-amplified from the respective KanMX-marked deletion strains in the BY4741 background (MATa his31 leu20 met150 ura30), obtained from Open Biosystems. The fragment encompassing NUP49-GFP fusion and HIS3 (Huh et al. 2003) was amplified from the Invitrogen strain collection. Integrations were verified by PCR and/or Southern blotting.
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).
Plasmid constructs and oligonucleotide primers
The primers used in this study are listed in Table 3. Plasmid pDB700 was designed for integrating the TDH3-GFP-RZ reporter marked by nureseothricin resistance marker gene NatMX (Goldstein and McCusker 1999) into the genomic trp1 locus. To this end, NatMX was amplified with oDB1082/1083 and cloned into the AatII site of the pRS304/2µ bearing TDH3-GFP-RZ (Dower et al. 2004), thus replacing its 2µ origin of replication. Integration was conducted after linearization at the Bsu36I site within the TRP1 gene sequence.
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) were designed for inserting the GAL-GFP-RZ and GAL-GFP-pA, respectively, by the gamma-integration method (Sikorski and Hieter 1989) into the intergenic region between ECM32 and BMH1, after linearization with NotI and selecting for Trp+ transformants. To generate pDB729, WT genomic SUS1 fragment was amplified by PCR with oDB1152 and oDB1153, cloned into the Not site of pDB700, followed by releasing the TDH-GFP-RZ reporter from the polylinker after digesting with SpeI + ApaI, repairing the ends with T4 DNA polymerase, and religating. Integration was conducted into TRP1 locus after linearization with Bsu36I, with selection for nurseothricin resistance.
Microscopy
GFP fusion proteins were observed in cells grown to OD 
0.5 and fixed for 15 min in 4% paraformaldehyde (without acetic acid) using an Olympus IX70-based DeltaVision workstation (Applied Precision). Z-stacks were taken at 0.2 µm step size and subjected to constrained iterative deconvolution. Positions of the TetR-GFP marked locus were scored in the z-section that cuts through the middle of the nucleus as described (Brickner and Walter 2004) into intranuclear, peripheral, and subperipheral (i.e., locus touching the nuclear envelope but not coplanar with it). The subperipheral fraction varied little in all conditions and therefore is not reported in Figure 3. Each experiment was done in replicate, and between 100 and 150 cells were scored per sample per time point in each replicate in all experiments shown. FISH with Cy3-labeled oligonucleotide probes was carried out according to Dower et al. (2004). Red/green channel signal offset due to chromatic aberration alone, as estimated by imaging TetraSpec beads (100 nm diameter, Invitrogen/Molecular Probes) under identical conditions, was negligible compared to the separation of FISH (Cy3) and GFP signals.
Chromatin immunoprecipitation and RNA analyses
RNAP II ChIP was performed using monoclonal antibody 8WG16 (Covance). Target DNA levels in input and IP samples were determined by real-time PCR using RotorGene (Corbett Research), and results were normalized as described (Abruzzi et al. 2004). The Northern hybridization signals in Figure 3 obtained with a GFP-specific probe were normalized to respective signals for SCR1, a RNAP III transcript, using ImageQuant software. Signal ratios were identical to the ChIP signal ratios in the respective strains.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.764108.
Received August 9, 2007; accepted September 21, 2007.
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REFERENCES |
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