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• W1992040206 abstract "Article31 August 2006free access 3′-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation Katharine C Abruzzi Katharine C Abruzzi Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Search for more papers by this author Dmitry A Belostotsky Dmitry A Belostotsky Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA Search for more papers by this author Julia A Chekanova Julia A Chekanova Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA Search for more papers by this author Ken Dower Ken Dower Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Search for more papers by this author Michael Rosbash Corresponding Author Michael Rosbash Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA Search for more papers by this author Katharine C Abruzzi Katharine C Abruzzi Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Search for more papers by this author Dmitry A Belostotsky Dmitry A Belostotsky Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA Search for more papers by this author Julia A Chekanova Julia A Chekanova Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA Search for more papers by this author Ken Dower Ken Dower Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Search for more papers by this author Michael Rosbash Corresponding Author Michael Rosbash Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA Search for more papers by this author Author Information Katharine C Abruzzi1, Dmitry A Belostotsky1,2, Julia A Chekanova1,2, Ken Dower1 and Michael Rosbash 1,2 1Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, MA, USA 2Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA *Corresponding author. Department of Biology, Howard Hughes Medical Institute, Brandeis University, 415 South Street, Waltham, MA 02454, USA. Tel.: +1 781 736 3160; Fax: +1 781 736 3164; E-mail: [email protected] The EMBO Journal (2006)25:4253-4262https://doi.org/10.1038/sj.emboj.7601305 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3′-end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene–nuclear periphery tether. Introduction mRNA export from the eukaryotic nucleus to the cytoplasm is preceded by multiple nuclear processing steps, many of which occur co-transcriptionally. Among these are three categories of covalent RNA modifications: addition of a 7′-methyl guanosine cap to the 5′-end, splicing, and addition of a 3′ stretch of adenosines (the poly-A tail). Nascent transcripts are also subject to numerous noncovalent processing events, that is, co-transcriptional recruitment of protein complexes that mediate these covalent alterations as well as proteins that contribute to messenger ribonucleoprotein particle (mRNP) export. Several mRNA export factors, including Npl3p, Sub2p and Yra1p, are recruited co-transcriptionally, whereas others such as Mex67p appear to associate with mRNP predominantly post-transcriptionally (Lei et al, 2001; Strasser et al, 2002; Zenklusen et al, 2002; Kim et al, 2004). Mex67p as well as other mRNP factors bind directly to nuclear pore components and facilitate mRNA egress to the cytoplasm (Segref et al, 1997; Strasser et al, 2000). Much of our knowledge about mRNA export and its relationship to other processing steps comes from experiments utilizing yeast temperature-sensitive strains. Using this strategy, multiple factors have been identified that are required to promote efficient export and avoid substantial poly-A RNA accumulation within the nucleus. These include early factors such as those involved in 3′-end cleavage and polyadenylation (pA), and late factors such as nuclear pore proteins and mRNP export factors (Amberg et al, 1993; Gorsch et al, 1995; Murphy and Wente, 1996; Santos-Rosa et al, 1998; Snay-Hodge et al, 1998; Hurt et al, 2000; Sträßer and Hurt, 2000; Hammell et al, 2002). For example, an mRNA export factor mutant (yra1-1) results in retention of poly-A RNA in the nucleus that is visualized by fluorescent in situ hybridization (FISH) with an oligo-dT probe (Sträßer and Hurt, 2000). Surprisingly, similar experiments using gene-specific FISH probes revealed that individual mRNAs are retained in a discrete nuclear focus or ‘dot’ under non-permissive conditions (Jensen et al, 2001). Furthermore, the number of dots correlates with the number of active genes, that is, one and two dots in haploid and diploid yeast strains, respectively (Jensen et al, 2001), and they appear similar to higher eukaryotic RNA foci that appear when RNA processing is impaired (Custodio et al, 1999). Some yeast dots appear related to a surveillance pathway that monitors mRNP quality, presumably to minimize nuclear export of faulty mRNPs (Hilleren et al, 2001). A number of studies indicate that surveillance and retention occur near the site of transcription (Thomsen et al, 2003; Dower et al, 2004), but the exact spatial and temporal relationship of actively transcribed genes and mRNP retention sites has never been addressed. Another potentially export-relevant connection between subnuclear compartments is that some highly expressed genes localize to the nuclear periphery upon transcriptional induction. These include the galactose-inducible GAL genes, mating pheromone inducible genes, INOI, HXK1 and genes that are responsive to the transcription factor Rap1p (Brickner and Walter, 2004; Casolari et al, 2004, 2005; Menon et al, 2005; Cabal et al, 2006; Taddei et al, 2006). Recent studies indicate that the activated GAL locus moves to the nuclear periphery and dynamically interacts with nuclear pores, which effects a sliding along the nuclear membrane (Cabal et al, 2006). Components of the SAGA histone acetylation complex (Sus1p and Ada1p), an mRNA export factor (Sac3p) and a nuclear pore component (Nup1p) are required for GAL gene localization to the nuclear periphery (Cabal et al, 2006; Drubin et al, 2006). These observations may indicate that transcription and mRNA export from some genes are directly coupled: newly transcribed mRNA from a pore-adjacent gene is ideally positioned for nuclear export as initially proposed by Blobel (1985) in the gene-gating hypothesis. A complication with the use of temperature-sensitive yeast strains for localization studies is that their interpretation can be complicated by indirect effects as well as the requisite use of heat-shock conditions, which may independently influence RNA export (Saavedra et al, 1997; Krebber et al, 1999). For these reasons, we previously carried out experiments to study export-related events in wild-type (WT) strain backgrounds. A set of high-copy reporter plasmids was used, which contain a GFP ORF under control of the strong TDH3 or GAL1 promoter, with 3′-end formation directed either by cleavage and pA signals from the corresponding gene (TDHpA or GALpA) or by a cis-acting hammerhead ribozyme element (RZ). The strategy was based in part on previous studies from our laboratory and others, which showed that a hammerhead ribozyme can cleave co-transcriptionally to generate an artificial 3′-end with no poly-A tail in vivo (Duvel et al, 2002; Dower et al, 2004; Lacadie et al, 2006). RZ-terminated RNAs (pTDH-GFP-RZ and pGAL-GFP-RZ) showed punctate nuclear staining by FISH, suggesting impaired RNA export and multiple nuclear accumulation sites from these high-copy plasmids. In contrast, pTDH-GFP-TDHpA and pGAL-GFP-GALpA-containing cells showed intense, uniform cell staining with only a hint of dots in the case of pGAL-GFP-GALpA (Dower et al, 2004). To investigate the relationship between genes, transcripts, 3′-end formation and the nuclear periphery in more detail and with better resolution, we constructed strains containing these reporter genes, all integrated at the same chromosomal location. Although the FISH results were similar to those observed with high-copy plasmid reporters including the lack of an Rrp6p effect (Dower et al, 2004; see below), we were now able to clearly detect a single nuclear dot with the GAL-GFP-GALpA gene. Further characterization also indicated that GAL-GFP-GALpA as well as the GAL-GFP-RZ dot consisted of non-nascent RNA, which is near but not coincident with the gene and persists after transcriptional shutoff. As GAL genes become more rim-associated upon transcriptional induction, we verified that our GAL1 promoter-driven reporter genes localize to the nuclear periphery in a manner indistinguishable from what has been described for natural GAL genes. Surprisingly, both the GAL-GFP-GALpA and GAL-GFP-RZ genes dissociate from the periphery well after transcriptional repression effected by glucose addition, indicating that ongoing transcription is not necessary to maintain a link between GAL genes and the nuclear periphery. As the kinetics of RNA dot disappearance parallels gene departure from the periphery, our experiments suggest that the mRNPs within the dot contributes to gene tethering at the nuclear periphery. Results To enable a comparison with our previous work utilizing high-copy (2μ) plasmids (Dower et al, 2004), we generated integrating constructs that express GFP under the control of two strong promoters: the regulated GAL1 promoter and the constitutive TDH3 promoter (Figure 1A). Transcript 3′-ends were directed by WT pA signals (e.g., GAL-GFP-GALpA), or by a self-cleaving hammerhead RZ (e.g., GAL-GFP-RZ). In this initial set of experiments, all constructs were integrated into the TRP1 locus of haploid Saccharomyces cerevisiae, and all strains contained single integrants as determined by both Southern blot analysis and PCR (data not shown). As previously shown, GFP mRNAs from reporters containing a pA signal (TDHpA or GALpA) have poly-A tails but the ribozyme terminated constructs do not (Dower et al, 2004 and data not shown). Figure 1.FISH of cells expressing GFP reporter constructs. (A) Diagram of four GFP reporter constructs used in this study. All constructs contain the GFP open reading frame under the control of either the constitutive TDH3 promoter or the inducible GAL1 promoter. The 3′-end of the constructs has either the TDH3 or GAL1 3′UTR or a self-cleaving hammerhead ribozyme (RZ). The position of the GFP FISH probes is shown. (B) FISH with a GFP-specific probe shows that GFP mRNA with a ribozyme generated 3′-end are retained as dots within the nucleus (TDH-GFP-RZ and GAL-GFP-RZ). In contrast, TDH3-promoted GFP RNAs that contain a TDH3 pA signal are diffusely localized throughout the cell (TDH-GFP-TDHpA). Surprisingly, GAL1-promoted GFP mRNAs that are polyadenylated via a GAL1 pA signal are retained dots within the nucleus (GAL-GFP-GALpA). Download figure Download PowerPoint RNA localization was assayed by FISH with antisense probes to GFP, and results were largely consistent with those previously reported for the 2μ plasmid reporters (Dower et al, 2004). The TDH3 promoter-driven gene with a WT TDH3 pA signal (TDH-GFP-TDHpA) showed cytoplasmic signal indicative of normal export and cytoplasmic stability, whereas the presence of an RZ 3′-end signal (TDH-GFP-RZ) gave rise to a single strong nuclear dot (Figure 1B). The RZ 3′-end also causes a slightly weaker cytoplasmic signal due to decreased RNA stability as well as nuclear processing difficulties (Dower et al, 2004). Similar results were obtained with the GAL promoter-driven genes, except that a nuclear dot was visible even with a WT GAL1 pA sequence (Figure 1B). The observation that GAL-GFP-GALpA mRNAs are retained in a dot, but TDH-GFP-TDHpA mRNAs are not, did not distinguish between a promoter effect or a 3′-end formation effect or both. To address this issue, we analyzed two additional integrated chimeric constructs with swapped promoters (Figure 2A): one contained the GAL1 promoter and the TDH3 pA signal (GAL-GFP-TDHpA), and the other contained the TDH3 promoter and the GAL1 pA signal (TDH-GFP-GALpA). Because the TDH-GFP-GALpA strain had a dot in both glucose and galactose, whereas the GAL-GFP-TDHpA strain had no expression in glucose and only diffuse cytoplasmic staining in galactose (Figure 2), this assigns the difference to the 3′-end signals (see Discussion). We conclude that both RZ and GALpA 3′-end signals generate a nuclear dot in an otherwise WT strain and therefore focused most subsequent experiments on a comparison of these two genes, GAL-GFP-RZ and GAL-GFP-GALpA. Figure 2.Dot formation is dependent on the nature of the 3′UTR. (A) Diagram of the two chimeric GFP reporter constructs. These constructs contain the GFP open reading frame and have either a TDH3 promoter and a GAL1 3′UTR (TDH-GFP-GALpA) or a GAL1 promoter and a TDH3 3′UTR (GAL-GFP-TDHpA). The position of the GFP FISH probes is shown. (B) FISH with a GFP-specific probe (red) and DAPI staining (blue) of the DNA was performed on TDH-GFP-GALpA and GAL-GFP-TDHpA cells grown either in glucose (GLU) or galactose (GAL). TDH-GFP-GALpA mRNAs are expressed and retained in dots in the nucleus when cells are grown in either glucose or galactose. GAL-GFP-TDHpA mRNAs are not expressed in glucose and in galactose they are diffusely localized throughout the cell. Download figure Download PowerPoint GAL-GFP-GALpA and GAL-GFP-RZ dots are independent of Rrp6p Previous studies showed that specific mRNAs are retained in nuclear dots when mRNA processing or export is impaired by shifting a number of conditional mutants to the non-permissive temperature (37°C; Amberg et al, 1993; Gorsch et al, 1995; Murphy and Wente, 1996; Santos-Rosa et al, 1998; Snay-Hodge et al, 1998; Hurt et al, 2000; Sträßer and Hurt, 2000; Hammell et al, 2002). This nuclear retention is dependent on the nuclear exosome component Rrp6p (Hilleren et al, 2001; Thomsen et al, 2003; Lund and Guthrie, 2005). In contrast, both GAL-GFP-GALpA and GAL-GFP-RZ dots are unaffected by a deletion of RRP6 (Figure 3). This result cannot be attributed to temperature; the GAL-GFP-GALpA, Δrrp6 and GAL-GFP-RZ, Δrrp6 strains still form dots even under heat-shock conditions (data not shown). This distinction suggests that not all dots are equivalent. Figure 3.GAL-GFP-GALpA and GAL-GFP-RZ dots are independent of the nuclear exosome component, Rrp6p. Cells expressing integrated GAL-GFP-GALpA or GAL-GFP-RZ in either a WT or Δrrp6 background were grown in galactose and message specific FISH was performed using oligos specific to GFP (see Figures 1 and 2). The ability of GAL-GFP-GALpA and GAL-GFP-RZ dots to form is not dependent on Rrp6p. Download figure Download PowerPoint mRNA in GAL-GFP-GALpA and GAL-GFP-RZ dots is post-transcriptional Previous results suggested that nuclear dots were at or near their genes of origin but did not discriminate between these two possibilities (Thomsen et al, 2003; Dower et al, 2004). The difference is important, as ‘near’ but not ‘at’ indicates that dots contain a substantial fraction of non-nascent RNA. This is especially relevant for GAL-GFP-GALpA dots; the normal 3′-end signal of this construct might indicate that an in situ signal reflects predominantly nascent RNA. To address this issue and to localize the gene simultaneously, we integrated GAL-GFP-RZ and a slightly modified GAL-GFP-GALpA (GFPkD200pA; the GFP fluorophore was mutated to generate a non-fluorescent GFP) at a different location, 5 kb from a pre-existing tandem TetO array at the BMH1 gene (Ciosk et al, 1998; see Materials and methods for details). By co-expressing a TetR-GFP fusion, we could simultaneously monitor RNA dots (by FISH; Figure 4A, shown in red) and the GAL-GFP gene (marked by TetR-GFP tethered to the TetO array; Figure 4A, shown in green). Figure 4.GFP dots are adjacent to but not overlapping the site of transcription and persist in the absence of transcription. (A) The GFP reporter genes were integrated adjacent to a TetO operator array to which TetR-GFP binds. When transcription is activated by steady-state growth in galactose, the GFP dots (FISH; shown in red) are adjacent to but not overlapping the site of transcription (TetR-GFP; shown in green). At 60 min after transcription is repressed by the addition of glucose, the dots are still visible and maintain a similar spatial relationship with the DNA locus. (B) Cells expressing GAL-GFP-GALpA or GAL-GFP-RZ were grown in galactose and at time zero, glucose was added to repress transcription. Cells were harvested for ChIP prior to glucose addition, 30 and 60 min after glucose addition and from cells maintained in glucose. RNA PolII occupancy on GAL-GFP-GALpA (blue) and GAL-GFP-RZ (red) was monitored at each timepoint. After 30 min, PolII levels are similar to those observed when transcription is repressed in glucose. (C) FISH using a GFP-specific probe was performed on the same cells used in (B). The number of cells containing GFP dots was counted (see Materials and methods). Both GAL-GFP-GALpA (blue) and GAL-GFP-RZ (red) expressing cells contain dots after transcription has been repressed by the addition of glucose. The GAL-GFP-RZ dots are more stable than the GAL-GFP-GALpA dots. Download figure Download PowerPoint Dots from both integrated genes were always distinct from—albeit always adjacent to—the marked chromosomal locus (Figure 4A). Because TetraSpeck beads (Invitrogen) examined under the identical conditions showed completely overlapping red/green images (data not shown; see Materials and methods), the dot–gene separation cannot be attributed to chromatic aberration. Given the 5 kB distance from the integrated constructs to the TetO array and the compaction ratio in yeast interphase chromatin (Bystricky et al, 2004), the non-overlapping signals therefore indicate that even the GAL-GFP-GALpA dots contain predominantly non-nascent RNA. The non-nascent nature of both dot RNAs was substantiated by the results of glucose chase experiments. Both reporter genes have a GAL1 promoter, which is rapidly inhibited by glucose addition (Mason and Struhl, 2005; see below), yet many dots are still easily visible by FISH after 60 min of growth in glucose (Figure 4A). Similar stability of nuclear mRNAs has been reported in mammalian systems (Weil et al, 2000). Moreover, both dots always remain adjacent to their genes, implying the existence of a persistent tether between mRNP within the dot and the locus of origin. To perform a more careful analysis of dot disappearance from GAL-GFP-GALpA and GAL-GFP-RZ strains, a quantitative glucose time course shutoff experiment was performed. The kinetics of dot disappearance was compared to the kinetics of RNA PolII clearance from the two GAL genes as assayed by chromatin immunoprecipitation (ChIP; Figure 4B). As expected (Mason and Struhl, 2005), transcription is inhibited by 30 min after glucose addition, and PolII occupancy at that time is comparable to steady-state glucose levels (no transcription). Indeed, 10 min is sufficient for a complete shutoff (data not shown). Although dot intensity does get weaker as a function of time after glucose addition (especially in the case of the GALpA-containing strain), dots persist much longer than expected for nascent RNA. Moreover, dot decay from the RZ gene is slower than from the GALpA gene. Forty-eight percent of GAL-GFP-RZ cells contain dots after 60 min in glucose, whereas only 27% of GAL-GFP-GALpA cells contain dots (Figure 4C). The differences in pA and RZ dot stability must reflect relative rates of GALpA and RZ RNA departure from dots after transcriptional repression, due to either turnover or export to the cytoplasm. The nature of the 3′UTR can alter co-transcriptional recruitment of an export factor To explore what might be responsible for the more persistent dots of the GAL-GFP-RZ gene, we compared the two genes by ChIP and assayed the association of PolII and two RNP proteins to different regions of nascent transcription complexes (5′, middle, 3′) under transcription-permissive (galactose) conditions. Both PolII and the RNP protein Sub2p were essentially indistinguishable across the genes, indicating that basic transcription and recruitment of at least some RNP proteins are unaffected by the RZ 3′-end (Figure 5B and C). However, GAL-GFP-RZ has higher levels of Yra1p at the 3′-end, showing that some feature of RNP protein recruitment or transfer to the nascent RNA is altered (Figure 5D). This suggests that the GAL-GFP-RZ mRNPs contain excess Yra1p in addition to lacking a pA tail (Duvel et al, 2002; Dower et al, 2004). mRNP differences more generally may be responsible for the differences in the dot stability from these two reporters. Figure 5.Co-transcriptional recruitment of mRNP proteins to GAL-GFP-GALpA and GAL-GFP-RZ. ChIPs were performed with RNA PolII and two different mRNP proteins (Sub2p and Yra1p) and the levels of each factor on GAL-GFP-GALpA and GAL-GFP-RZ was determined. (A) Diagram of the GFP reporters and the location of the PCR primers used in ChIP experiments. (B) RNA PolII recruitment is similar on GAL-GFP-GALpA and GAL-GFP-RZ. (C) The mRNP protein, Sub2p, is recruited similarly to both reporter genes. (D) Yra1p is enriched on the 3′-end of the GAL-GFP-RZ gene in comparison to the GAL-GFP-GALpA gene. Download figure Download PowerPoint GAL-GFP-GALpA and GAL-GFP-RZ associate with the nuclear periphery Because the GAL locus increases its association with the nuclear periphery in a transcription-dependent fashion, we addressed whether there might be a relationship between dots and intranuclear gene localization. We compared the nuclear localization of three different reporter constructs: GAL-GFP-GALpA (dot forming), GAL-GFP-RZ (dot forming) and GAL-GFP-TDHpA (non-dot forming). All three of these reporters were integrated into the genome adjacent to the Tet0-GFP array (see Materials and methods for details). To visualize the nuclear rim, Nup49p fused to CFP (NUP49-CFP; Shor et al, 2005) was expressed in the cells. Cells were fixed and visualized using a DeltaVisionTM (Applied Precision) optical sectioning microscope (see Materials and methods). The TetR-GFP mark was scored as internal (I), subperipheral (S; touching the nuclear rim from interior of nucleus) or peripheral (P; coincident with the rim), and a distribution among the three positions was calculated (Figure 6A). Figure 6.Intranuclear localization of the GAL-GFP-GALpA, GAL-GFP-RZ and GAL-GFP-TDHpA genes. The GAL reporter genes were integrated into the genome adjacent to a TetO array. TetR-GFP was co-expressed to visualize the gene and NUP49-CFP was expressed to mark the nuclear periphery. (A) The intranuclear localization of the gene (TetR-GFP) relative to the nuclear periphery was categorized as being internal (I), subperipheral (S; proximal to but not overlapping the nuclear membrane), or peripheral (P; adjacent to the nuclear membrane). Representative images of each category are shown. (B) The intranuclear localization of the GAL-GFP-GALpA, GAL-GFP-RZ and GAL-GFP-TDHpA genes was determined during transcriptionally active (GAL; galactose) or repressed (GLU; glucose) states. Both GAL-GFP-GALpA and GAL-GFP-RZ become more associated with the nuclear periphery when grown in galactose. In contrast, there was no substantial difference in the localization of the GAL-GFP-TDHpA gene in glucose compared to galactose. (C) The intranuclear localization of the GAL-GFP-RZ gene was examined 0, 15, 30 and 60 min after transcriptional shutoff by the addition of glucose as well as in steady-state glucose. The GAL-GFP-RZ gene is enriched in the nuclear periphery for up to 60 min after transcriptional shutoff suggesting that the GAL-GFP-RZ gene does not require active transcription to maintain its peripheral localization. (D) The intranuclear localization of the GAL-GFP-GALpA gene was examined 0, 15, 30 and 60 min after transcriptional shutoff by the addition of glucose as well as in steady-state glucose. The GAL-GFP-GALpA remains at the nuclear periphery for 30 min after transcriptional shutoff illustrating that the gene does not require active transcription to maintain its peripheral localization. However, the GAL-GFP-GALpA is released from the nuclear periphery more quickly than the GAL-GFP-RZ gene. Download figure Download PowerPoint Both the GAL-GFP-GALpA gene and the GAL-GFP-RZ gene show an enhanced peripheral localization when transcription of these genes is induced by growth in galactose (compare galactose to glucose; Figure 6B). The quantitative results are essentially identical to what has been reported previously for an endogenous GAL locus (Casolari et al, 2004; Cabal et al, 2006). In contrast to endogenous loci where multiple neighboring genes might undergo activation, these data indicate that activation of this single, added gene is sufficient for enhanced peripheral localization of the region. In contrast to GAL-GFP-GALpA, the GAL-GFP-TDHpA gene shows little change in nuclear localization. It has an intermediate phenotype on both carbon sources, that is, not as peripheral as the other two genes on galactose and not as central as on glucose (Figure 6B). This difference is not due to an alteration of transcriptional induction in galactose: RNA polymerase II (PolII) ChIPs indicate that this chimeric gene is induced similarly to GAL-GFP-GALpA (data not shown). This result suggests that the GAL1 promoter is insufficient to localize a gene to the nuclear periphery and indicate that the nature of the 3′UTR can impact gene localization. Studies to date show that transcriptional induction is required for the association between GAL genes and the nuclear periphery, but it is not clear whether continued transcription induction is necessary for its maintenance. For these reasons, we compared the intranuclear localization of GAL-GFP-GALpA and GAL-GFP-RZ after shifting from galactose to glucose (Figure 6C and D). Remarkably, the data indicate that the two genes maintain their peripheral nuclear localization for different amounts of time after glucose addition: the RZ gene maintains a peripheral localization much longer than the GALpA gene. Indeed, the intranuclear localization of the RZ gene may not change at all after 60 min in glucose (Figure 6C). In contrast, the GAL-GFP-GALpA gene becomes progressively more interior and less peripheral; by 60 min, localization is almost indistinguishable from steady-state glucose grown cells (Figure 6D). Nonetheless, even this gene has a more peripheral localization 30 min after transcription shutoff than under steady-state repressed conditions. As 30 min is sufficient to completely inhibit transcription from the GAL promoter (Figure 4B; data not shown; Mason and Struhl, 2005), this indicates that continued transcription is not necessary to maintain peripheral gene localization. Moreover, the relative locus dissociation kinetics (Figure 6C and D) resembles the relative dot decay time courses of the two genes in glucose (Figure 4C), further suggesting a relationship between mRNP in retained foci and peripheral gene localization. Endogenous GAL1 mRNA accumulates in the nucleus in dots that are independent of RRP6 The movement of the GAL locus to the nuclear periphery has been well documented in a number of studies (Casolari et al, 2004; Cabal et al, 2006; Schmid et al, 2006). If there is a general role of post-transcriptional mRNP in tethering active loci to the nuclear periphery, GAL mRNAs should accumulate in nuclear dots. Indeed, FISH with GAL1-specific probes show that GAL1 mRNAs accumulate in dots in both a WT and a Δrrp6 background (Figure 7). Furthermore, similar to the GAL reporter ge" @default.
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• W1992040206 title "3′-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation" @default.
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• W1992040206 doi "https://doi.org/10.1038/sj.emboj.7601305" @default.
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