![[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. 19, Issue 10, 4310-4318, October 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Centro Andaluz de Biologia Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC, 41092 Sevilla, Spain
Submitted April 7, 2008;
Revised July 18, 2008;
Accepted July 22, 2008
Monitoring Editor: Marvin P. Wickens
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Buratowski, 2005; Cole and Scarcelli, 2006). Incorrectly spliced or 3'-end processed transcripts are retained within the nucleus, providing evidence that mRNA maturation and export are linked to each other (Lei and Silver, 2002). Any failure in the formation of an export-proficient mRNP would cause nuclear RNA retention and could trigger nuclear mRNA decay in association with the nuclear pore complex (NPC; Galy et al., 2004) and with transcription (Andrulis et al., 2002). Importantly, recent reports in yeast, Drosophila and human lymphocytes have revealed that dynamically regulated genes are recruited to the nuclear periphery when transcription is activated (Casolari et al., 2004; Faria et al., 2006; Mendjan et al., 2006; Ragoczy et al., 2006; Taddei et al., 2006). It has been suggested that the activation of genes in the nucleus is linked to specialized NPCs that in turn facilitate efficient mRNA export (Blobel, 1985). Supporting this hypothesis, gene positioning at the proximity of the NPC has been shown to be dependent on factors involved in transcription initiation and mRNA export (Cabal et al., 2006; Dieppois et al., 2006; Kurshakova et al., 2007; Chekanova et al., 2008; Köhler et al., 2008).
mRNP formation seems to play a key role not only in gene expression but also in other cellular processes such as the maintenance of genome integrity. An example of this connection between mRNP formation and genetic integrity is provided by the THO complex of Saccharomyces cerevisiae, a conserved four-protein complex composed of stoichiometric amounts of Tho2, Hpr1, Mft1, and Thp2 (Chavez et al., 2000), which is recruited to active chromatin in vivo (Strasser et al., 2002; Zenklusen et al., 2002). Null mutations of any component of THO lead to similar phenotypes of transcription impairment and RNA export defects (Chavez et al., 2000; Strasser et al., 2002), the most intriguing phenotype being their transcription-associated hyper-recombination. Analysis of yeast THO mutants has led to the idea that transcription-associated recombination (TAR) may be a consequence of transcriptional-elongation impairment (Aguilera and Gomez-Gonzalez, 2008). One major cause of this phenomenon is the cotranscriptional formation of R-loops (DNA-RNA hybrids) formed behind the elongating RNAPII (Huertas and Aguilera, 2003). In the current view, the THO complex would participate in cotranscriptional formation of export-competent mRNPs during transcription elongation preventing R-loop formation. The observation that depletion of the ASF/SF2 splicing factor in chicken DT40 cells and human HeLa cells also lead to genomic instability linked to R-loop formation indicates that a number of mRNA-processing enzymes may contribute to prevent the formation RNA-dependent structures that may trigger genome instability (Li and Manley, 2005).
THO forms, together with the RNA export proteins Sub2/UAP56 and Yra1/Aly, a larger and conserved complex termed TREX (Strasser et al., 2002; Rehwinkel et al., 2004). Interestingly, yeast mutants of SUB2 and YRA1 are synthetic-lethal with THO mutations and also lead to hyper-recombination and gene expression defects (Fan et al., 2001; Jimeno et al., 2002; Strasser et al., 2002). Furthermore, mutations in the genes of the Mex67-Mtr2 export factor, the Nab2 hnRNP or the NPC-associated Thp1 and Sac3 proteins also confer hyper-recombination and gene expression defects (Gallardo and Aguilera, 2001; Jimeno et al., 2002; Gallardo et al., 2003), even though this is not a general feature of mRNA-processing mutations (Luna et al., 2005). Despite some similarities there are important differences between THO and Thp1 and Sac3. Thus, Sub2 overexpression suppresses THO mutants, but it inhibits growth of the Thp1 mutant. Also, Nab2 overexpression suppresses the Thp1 mutant but has no effect on THO mutants (Jimeno et al., 2002; Gallardo et al., 2003). Notably, in contrast to THO, Thp1 and Sac3 associate with nucleoporins at the nuclear basket and mediate export of mRNPs (Fischer et al., 2002; Lei et al., 2003). In addition, Thp1 and Sac3 are found in association with Cdc31 centrin (Gallardo et al., 2003; Fischer et al., 2004), which functions in the duplication of microtubule-organizing centers, and with Sus1, a small protein conserved from yeast to humans recently identified as a novel component of SAGA histone-modification complex involved in transcription initiation (Rodriguez-Navarro et al., 2004; Zhao et al., 2008). The observation that Sus1 is involved in the SAGA-dependent histone H2B deubiquitylation and maintenance of normal H3 methylation levels (Köhler et al., 2006) and that Thp1, Sac3, Sus1, and Ada2, a bona fide component of SAGA, act in the repositioning and dynamic motility of SAGA-dependent loci, to the nuclear periphery upon transcriptional activation (Cabal et al., 2006; Kurshakova et al., 2007; Chekanova et al., 2008) suggests the possibility that Sus1 could be a bridge protein between transcription, via SAGA, and mRNA export.
Given all these observations, the question emerging is how RNA export factors, such as Thp1 and Sac3 control genome integrity and whether they are functionally related to THO/TREX, which is physically bound to active chromatin and does not seem to be located at the nuclear periphery. Another emerging question is whether or not the main function of Sus1 is linked to transcription initiation as part of the SAGA complex. Here we show that Thp1, Sac3, and Sus1 form a functional unit with a role in transcription elongation that is independent of SAGA and is linked to RNA export. Our data reveal that the Thp1-Sac3-Sus1-Cdc31 (THSC) complex, together with THO/TREX, define a specific pathway connecting transcription elongation with nuclear export by an RNA-mediated dynamic process. This provides a feedback mechanism for the control of transcription that guarantees genetic stability of highly transcribed DNA regions.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
NS (Prado et al., 1997), pRS314L-lacZ, pRS314GL-lacZ (Piruat and Aguilera, 1998), pCM184-LAUR (Jimeno et al., 2002), pCM189-LEU2 (Gonzalez-Barrera et al., 2002), pGCYC1-402 (Rondon et al., 2003b), pGL-ribm, pGL-Rib+, pPHO5-ribm-lacZ, pPHO5-Rib+-lacZ, pGAL:RNH1 (Huertas and Aguilera, 2003) p416-GAL1 (Mumberg et al., 1994), and p413GALAID (Gomez-Gonzalez and Aguilera, 2007) were described previously. Plasmid Ptet-SUB2 containing the complete SUB2 coding sequence under control of the tetO promoter was constructed by inserting the 1.3-kb BamHI SUB2 fragment obtained by PCR using the primers 5'-ATC GCG GAT CCA TGT CAC ACG AAG GTG AA-3' and 5'-CGC GCG GAT CCT TAA TTA TTC AAA TAA GT-3' into pCM189 (Gari et al., 1997).
Chromatin Immunoprecipitation
For chromatin immunoprecipitation (ChIP) experiments, strains were grown in synthetic complete medium (SC) 2% glycerol-2% lactate to an OD660 of 0.5. The culture was split in two, and one-half was supplemented with 2% glucose (repressed transcription) and the other with 2% galactose (activated transcription). Samples were taken after 4 h, and ChIP assays were performed as described (Hecht and Grunstein, 1999). Monoclonal anti-Rpb1-CTD antibody 8WG16 (Berkeley Antibody Company, Richmond, CA) and protein A-Sepharose were used for RNAPII immunoprecipitation. The GFX purification system (Amersham, Indianapolis, IN) was used for the last DNA purification step. We used the PCR of the intergenic region at positions 9716–9863 of chromosome V as a negative control. Real-time quantitative PCR and calculations of the relative abundance of each DNA fragment were performed as described (Huertas et al., 2006).
In Vitro Transcription Elongation Assays
Transcription elongation was assayed in yeast whole cell extracts (WCEs) in vitro. WCEs were prepared from yeast cells grown in rich medium YEPD at 30°C to an OD600 of 1, and the reactions were carried as described previously (Rondon et al., 2003b).
Recombination and Mutation Analysis
Recombination and mutation frequencies of the monocopy centromeric plasmids pRS316L, pRS316LYNS, pRS314GL-lacZ, pRS314L-lacZ, pGL-ribm, and pGL-Rib+ described earlier were obtained as the average of three to four median frequencies from two different transformants each and for each genotype tested. Median frequencies were obtained as previously described (Santos-Rosa and Aguilera, 1994) from six independent colonies per transformant.
Miscellaneous
Northern analyses were performed according to standard procedures with 32P-radiolabeled probes. Probes used were described previously (Chavez et al., 2000). RNA analyses for the Rib+ and ribm constructs were performed as described in Huertas and Aguilera (2003).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cells (Gallardo et al., 2003). Here we used this feature to obtain new insights into the functional relationship of Thp1 and Sac3 and other proteins involved in mRNP biogenesis. We placed the SUB2 gene in a centromeric plasmid under the control of the Tet promoter, which allowed the expression of the gene in the absence of doxycycline (Ptet-SUB2), and transformed different mutants of mRNP biogenesis factors (Figure 1A). Figure 1B shows that growth of mutants of the THSC complex as sac3 and sus1 were affected when SUB2 was overexpressed. The results suggest that Thp1, Sac3, and Sus1 act as a unit that functionally interacts with Sub2. We also analyzed two cdc31 mutants, (cdc31-1 and cdc31-115); cdc31-1 is defective in spindle pole body (SPB) duplication, cell integrity and morphogenesis (Sullivan et al., 1998), whereas cdc31-115 is not affected in SPB duplication (Ivanovska and Rose 2001) but has an mRNA export defect (Fischer et al., 2004). Overexpression of Sub2 did not have any effect on neither of these mutants.
|
, rat1-1, and mtr4-1), and mRNA export (mex67-5, yra1-1, nab2-1, gle1-4, and rat8-2). Nevertheless, it had no effect in mutants of other nuclear processes, such as protein transport (crm1-1) and the processing of other RNA species (dbp7) and in mutants in genes encoding mRNA binding proteins (npl3). Sub2 overexpression inhibited growth of a number of mutants including those in nucleoporins that interact with Thp1 and Sac3 (nup60) and in nucleoporins of the Nup84 complex (nup84, nup133, and nup120), but not of other nucleoporins (nup2, nup100, nup188, and nup170; Figure 1C). In addition, Sub2 overexpression inhibits growth of a mutant of Nab2, an hnRNP (heterogeneous nuclear ribonucleoprotein) that interacts genetically with Thp1 (Gallardo et al., 2003; Figure 1C). Therefore, our results suggest that the growth-defect phenotype caused by overexpression of SUB2 is specific to a subset of mRNP biogenesis and export factors that could define a particular pathway.
Because Sus1 was identified as part of the SAGA histone acetylase complex (Rodriguez-Navarro et al., 2004) and Sub2 overexpression inhibited growth of sus1 mutants, we asked whether Sub2 overexpression also affected SAGA mutants. Notably, we did not observe growth inhibition in mutants of different representative genes of the functional and structural modules of SAGA (Figure 1D). This suggests that despite the association of Sus1 with SAGA, a function of Sus1 is directly related to Thp1 and Sac3 in mRNP biogenesis and export rather than SAGA.
sus1 But Not SAGA Mutants Confers Transcription-dependent Hyper-Recombination
As SUS1 and CDC31 encode proteins that have been shown to copurify with Thp1 and Sac3 (Fischer et al., 2002; Gallardo et al., 2003; Rodriguez-Navarro et al., 2004), we wondered whether their mutations also lead to increased TAR. Representative mutants of the different modules of SAGA were included in the study. For the analysis of transcription-dependent recombination, we used the plasmid-based system LYNS based on 0.6-kb leu2 repeats in which transcription has to proceed through a long and GC-rich intervening sequence. In this system thp1 and sac3 lead to an increase in recombination of two- to three orders of magnitude above wild-type levels (Gallardo and Aguilera, 2001; Gallardo et al., 2003). As control, we used the L system, identical to LYNS but without intervening sequences between the leu2 repeats and which is not significantly affected by thp1. Here we show that whereas sus1 mutant showed a clear increase in recombination in LYNS (5.5-fold) but no effect in the L system, ubp8, sgf11, spt7, spt8, gcn5, and spt20 mutants showed low recombination levels in both the L and LYNS systems (Figure 2A). Besides, none of the Cdc31 centrin mutants analyzed (cdc31-1 and cdc31-115) showed hyper-recombination, neither at 30°C (Figure 2A) nor at restrictive temperature 34°C (data not shown). Consistent with Sub2 overexpression data (Figure 1), these results suggest that Sus1 share functions with Thp1 and Sac3 in the maintenance of genetic integrity. Although we found no hyper-recombination in the cdc31 mutants tested, whether or not Cdc31 has a related or more distant role to the other subunits of the THSC complex would need to be addressed with specifically selected cdc31 alleles, given that Cdc31 is the only essential subunit of this complex.
|
is transcription-dependent. To demonstrate this, we determined the effect of sus1 on recombination in the L-lacZ and GL-lacZ systems carrying 0.6-kb leu2 direct repeats flanking the lacZ open reading frame under conditions of low (GAL1 promoter in 2% glucose), medium (LEU2 promoter), and high levels of transcription (GAL1 promoter in 2% galactose). As can be seen in Figure 2B and Supplemental Figure S1, the higher the levels of transcription the stronger the increase in recombination. The results demonstrate that hyper-recombination in sus1 is mainly transcription dependent as has been described for thp1 and sac3 mutants.
Transcription Elongation Is Impaired in THSC Mutants In Vivo, But Only Slightly In Vitro
We have previously reported that thp1 and sac3 mutants are defective in transcription through high G+C content genes like lacZ (Gallardo et al., 2003). To test whether this is also the case of sus1, we analyzed gene expression in the LAUR expression system (Jimeno et al., 2002) containing a 4.15-kb lacZ-URA3 translational fusion under the control of the Tet promoter. As can be seen in Figure 3A, sus1 and sac3 cells, carrying the LAUR system, were unable to form colonies on synthetic complete medium lacking uracil (SC-Ura-Trp), indicating that they did not express the lacZ-URA3 fusion. Consistently, they did not produce β-galactosidase activity (data not shown). Northern analysis shows that whereas wild-type cells could express this construct properly, sus1 and sac3 mutants showed a reduction in mRNA accumulation (Figure 3B), similar to that observed for thp1 (Gallardo et al., 2003). Such a reduction was not caused by an impairment of transcription initiation at the Tet promoter because mRNA accumulation of the LEU2 gene under the Tet promoter (pCM189-LEU2 expression system) was the same in sus1 and sac3 as in the wild type (Figure 3B). We conclude, therefore, that sus1 lead to similar gene-expression defects as those of thp1 and sac3 mutants.
|
, thp1, and sac3 suggest that transcription elongation may be impaired, as was previously shown for THO mutants (Rondon et al., 2003b). To determine whether THSC mutants were impaired in transcription elongation we used our previously reported in vitro system containing two G-less cassettes (Rondon et al., 2003a), in which transcription-elongation efficiency is determined in whole cell extracts (WCEs) by the values of the ratio of accumulation of the downstream (376-nt long) versus the upstream (84-nt long) G-less RNA fragments (Figure 4; see Materials and Methods). We assayed in vitro transcription elongation in thp1, sac3, and sus1. In addition, we included in our analysis two mutants of SAGA: ada2, impaired in histone acetylation, and ubp8, mutated in the Ubp8-Sgf11 deubiquitinylating enzyme, shown to control binding of Sus1 to SAGA (Köhler et al., 2006), because recently both have been reported to be linked to transcription elongation (Govind et al., 2007; Wyce et al., 2007). As can be seen in Figure 4, the transcription-elongation efficiencies of thp1, sac3, sus1, ada2, and ubp8 were above 80% of the wild-type values, in some cases close to wild-type levels, whereas in the THO mutants used as controls (tho2 and hpr1) efficiencies were 60% or lower. As transcription is coupled with mRNA export and THSC is located at the nuclear periphery in association with the NPC, it is possible that the effect of the THSC complex on transcription is relevant when coupled to the NPC, and not in cell extracts in which coupling is disrupted.
|
). RNAPII occupancy was determined at a 5' and a 3' region of YLR454w in thp1 and sac3 mutants, using hpr1 as a control. Figure 5 shows that the presence of RNAPII at the 3'-end of the gene was reduced with respect to the 5'-end to 51 and 73% in thp1 and sac3 mutants, respectively. These values were similar to those of hpr1, indicating that the RNAPII elongation is decreased in THSC mutants in vivo.
|
). To determine whether this was also the case for THSC and also Sub2 mutants, this last component of the TREX complex, we analyzed transcription in the previously described Rib+ and ribm constructs (Figure 6A, Supplemental Figure S2), in which a PHO5-Rib-lacZ transcriptional fusion containing either a wild-type (Rib+) or a mutated (ribm) hammerhead ribozyme sequence was placed under the control of the GAL1 promoter (Huertas and Aguilera, 2003). In both constructs, a 2.2-kb-long mRNA is synthesized, but in the Rib+ construct the active hammerhead ribozyme cleaves the transcript, leading only to a short 0.6-kb-long mRNA fragment downstream of the ribozyme. We analyzed transcription in thp1, used as representative mutant of the THSC complex, and in sub2, hpr1, and tho2 as representative mutants of THO/TREX. Northern analyses revealed that the thp1 mutant was suppressed in the Rib+ construct, as it was the case for hpr1, tho2, and sub2 mutants. In such mutants only 30–40% of the transcription efficiency of the wild type can be observed because this is the maximum level of transcription reached with these types of constructs (Garcia-Rubio et al., 2008).
|
strains was obtained when the ribozyme was active (GL-Rib+) in the presence of highly expressed RNAse H that would remove the RNA chain of a putatively formed R-loop (Huertas and Aguilera, 2003). Here we show that hpr1, tho2, sub2, and thp1 strains carrying the GL-Rib+ construct and overexpressing RNaseH1 had significantly reduced recombination frequencies compared with those of GL-ribm, as was previously shown for hpr1 mutants (Huertas and Aguilera, 2003). Altogether the results suggest that not only in THO complex, as shown with hpr1 and tho2 mutants, but also in TREX, as shown with sub2, and THSC, as shown with thp1, both the transcription impairment and hyper-recombination are dependent on the nascent mRNA.
THSC Inactivation Strongly Enhances the Mutator Ability of Human Activation-induced Cytidine Deaminase Protein
Activation-induced cytidine deaminase (AID) is a specific B-cell enzyme essential for immunoglobulin (Ig) somatic hypermutation and class switching that acts in vitro on single-stranded DNA, one of its in vivo targets being the S regions of Ig genes, in which R-loops are formed (Muramatsu et al., 2000; Revy et al., 2000; Okazaki et al., 2002). We have recently reported that the heterologous overexpression of human AID is able to strongly induce both mutation and recombination in yeast THO mutants (Gomez-Gonzalez and Aguilera, 2007). This is explained by the fact that R loops formed in THO mutants leave the nontranscribed chain (NTS) as single-strand DNA (ssDNA), thereby increasing accessibility to AID. Consistently, in mft1 cells expressing AID, mutations were 10-fold higher in the NTS, whereas such a strand bias was not observed in the wild type (Gomez-Gonzalez and Aguilera, 2007).
We wondered, therefore, whether THSC inactivation by thp1, stimulated the action of AID, as an indirect manner to assess whether R-loops also formed in THSC mutants. We used the LAUR system. In this assay Ura– colonies are selected in SC-FOA. As can be seen in Figure 7, AID expression increases the frequency of Ura– colonies fivefold in wild-type cells and 19-fold in mft1 cells, consistent with previously reported data (Gomez-Gonzalez and Aguilera, 2007). Noteworthy, the effect of AID was not specific to THO mutants but was also observed in thp1 mutant, in which AID increased mutations 58-fold. Such an increase was not seen in spt4 strains and others mutants in factors involved in transcription such as Spt6 and Rpb2 (data not shown). Therefore, we can conclude that in THSC mutants, there is a transcription-dependent transient accumulation of ssDNA that facilitates AID action. This is consistent with the presence of R-loops that would leave the nontranscribed chain as single-stranded, as in THO mutants (Gómez-González and Aguilera 2007).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
THO contributes to the formation of an optimal mRNP, presumably facilitating the assembly of RNA-binding proteins onto the nascent mRNA such Sub2 or Yra1. A defective THO complex would lead to failure in this process that in turn would create suboptimal mRNP that would not be competent for export and would contribute to inhibit transcription elongation and to trigger recombination, with the concomitant formation of an R loop (Huertas and Aguilera, 2003). Here we show that THO is not the only complex in the absence of which, failures of transcription-RNA export coupling causes genome instability. Hyper-recombination and mRNA accumulation defects were previously observed in mutants of Sub2, Mex67, Thp1, and Sac3 (Jimeno et al., 2002; Gallardo et al., 2003), but whether mutations in these genes led to transcription-elongation impairment in an RNA-dependent manner was not known.
It is worth noticing that Sus1 is part of two different protein complexes, THSC and SAGA, and has been proposed to act as a bridge between mRNA export and transcription (Rodriguez-Navarro et al., 2004). Recent data suggest that Sus1 could function in histone acetylation and transcription in a SAGA-dependent manner and is necessary for RNA export (Köhler et al., 2006; Zhao et al., 2008). Nevertheless, our results suggest that Sus1 plays an important role as part of the THSC complex in RNA biogenesis/export. This is deduced from the observations that overexpression of Sub2 inhibited the growth of sus1 cells, because thp1 and sac3 and other RNA export factor mutants, such as pap1, mex67, yra1, nab2, gle1,and dbp5, and nucleoporin mutants such as nup60, nup84, nup133, and nup120, but not any of the SAGA mutants tested, including ubp8 and sgf11, encoding the closest partners of Sus1 in the SAGA complex (Köhler et al., 2006) and sgf73, mutated in the Sgf73 subunit that mediate recruitment of Thp1-Sac3 to SAGA (Köhler et al., 2008). It is likely that overexpression of Sub2 leads to an aberrant mRNP structure causing an irreversible block of mRNP biogenesis and export and hence growth inhibition in THSC mutants deficient in RNA export. Besides, sus1 confers a reduction in mRNA accumulation of lacZ similar to thp1 and sac3, whereas this transcription defect is not observed in ubp8 and sgf11 mutants, the two SAGA subunits functionally linked to Sus1 (data not shown). Furthermore, sus1 mutants share the in vivo transcription impairment phenotype and transcription-dependent hyper-recombination of thp1 and sac3 mutants; whereas SAGA mutants show wild-type recombination phenotypes (Figure 2A and Supplemental Figure S3). Altogether, these data suggest that Sus1 forms a functional unit with Thp1 and Sac3 (THSC) with a role in mRNP biogenesis independent of SAGA. Nevertheless, and as it happens with some subunits of other protein complexes, such as Tex1 of TREX or Mft1 of THO (Luna et al., 2005), the relevance of Sus1 in THSC seems to be lower than that of Thp1 and Sac3, according to the milder phenotypes of sus1 mutants. This would be in agreement with the recent work on Sgf73 and the THSC complex that indicates that this factor mediates the recruitment of Thp1 and Sac3 to SAGA and their stable interaction with Sus1-Cdc31 (Köhler et al., 2008).
The similarity of transcription and recombination phenotypes of THSC mutants with those of THO and the observation that THSC plays a role in maintaining the nuclear pore localization of genes (Cabal et al., 2006, 2008; Kurshakova et al., 2007) opens up the possibility that THSC could also bind to active chromatin in a transcription-dependent manner. Nevertheless, so far we have been unable to show that Thp1-Sac3 is recruited to active chromatin (data not shown). The observation that THSC mutants have a weak effect on transcription elongation in vitro compared with THO mutants is consistent with a role of THSC in mRNP biogenesis that would be coupled to its function at the nuclear pore. Our in vitro assays have been performed with WCEs in which nuclear envelops are disrupted and the DNA substrate is added independently and apart of NPCs. Concordantly, the effect of a complex that interacts with the nuclear pore as THSC (Fischer et al., 2002) may not be properly detected with this in vitro assay, but it can be observed in vivo assays performed with intact cells. So far binding of Sus1 to GAL1 promoter has been reported, suggesting that tethering of the DNA to the nuclear pore via THSC could be via promoters, regardless of its transcriptional state (Rodriguez-Navarro et al., 2004; Cabal et al., 2006; Kurshakova et al., 2007), but this binding does not explain its in vivo transcription-elongation impairment.
A key result to understand the specific transcription phenotypes of the mutants of this process is provided by the analysis of the effect in different mutations of THO subunits, Sub2 and Thp1, on ribozyme-containing transcription and recombination assays and on hyper-mutation caused by human AID. Our study reveals that mutants in these proteins lead to a DNA structure susceptible to the action of human AID, as was recently shown in THO mutants (Gomez-Gonzalez and Aguilera, 2007). This, together with the observation that AID acts preferentially on ssDNA (Chaudhuri et al., 2003) is consistent with formation of R-loops in these mutants, as has been shown for hpr1 (Huertas and Aguilera, 2003) and the S regions of Ig genes where AID acts (Yu et al., 2003). Therefore, in contrast to the idea that THO could be a unique factor acting at transcription sites with a role preventing the interaction of nascent RNA with the DNA, other factors acting downstream on mRNP biogenesis and export has similar effect. This implies a feedback mechanism by which improperly formed mRNPs, presumably stacked at the nuclear pore, have a backward effect promoting transcription impairment and genetic instability. It is possible that THSC-malfunction disrupts both RNA export and mRNP assembly, causing transcription elongation impairment via a mechanism similar to that occurring in THO mutants and has yet to be deciphered.
Alternative mechanisms can explain the peripheral location of activated genes. These may involve either promoter-interacting proteins such as SAGA components or mRNP biogenesis and processing factors such as the RNA export factor Mex67 or the Mlp1 factor involved in mRNA surveillance (Dieppois et al., 2006; Chekanova et al., 2008). Our data suggests a model in which the biogenesis of mRNPs ends in the localization of the transcribed DNA at the proximity of the NPC could be via the subsequent action of THO, Sub2-Yra1, Mex67-Mtr2, and THSC in a transcription- and RNA-mediated manner. This process would be independent of SAGA and would prevent the generation of suboptimal mRNPs that could react with DNA, compromising genome integrity. The recent observation that the Mex67 export factor is recruited to chromatin in a transcription and THO-dependent manner (Gwizdek et al., 2006) provides a new scenario in which Mex67 may also be loaded onto the mRNP during transcription to allow its subsequent export through the NPC. This direct connection may explain the transcription defects and hyper-recombination phenotype of mex67-5 mutants previously reported (Jimeno et al., 2002). Our results, therefore, define a specific pathway that controls the fate of the mRNA from the site of transcription to the nuclear pore as a key process in the maintenance of genome integrity. Presumably, these protein factors from THO to THSC may function on the nascent RNA in a dynamic process starting on the DNA and finishing up in close proximity to the NPC. To unravel why THSC has a feedback effect in transcription elongation and genome integrity will help understand how different nuclear processes are interconnected and the key function of these mRNP biogenesis and export factors in maintaining genome integrity.
|
ACKNOWLEDGMENTS |
|---|
at Heidelberg, Heidelberg, Germany) and M. Rose (Princeton University, Princeton, NJ) for yeast cdc31 mutants, S. Jimeno for helpful comments on the manuscript, and D. Haun for style supervision. This work was supported by grants from the Spanish Ministry of Science and Education (SAF2003-00204 and BFU2006-05260) and Junta de Andalucía (CVI102 and CVI624). C.G.-A. and B.G.-G. were the recipients of (Formación Profesorado Universitario) Ph.D. training grants from the Spanish Ministry of Science and Education. |
Footnotes |
|---|
* Present address: The Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, United Kingdom. 
Address correspondence to: Andrés Aguilera (aguilo{at}us.es)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aguilera, A., and Gomez-Gonzalez, B. (2008). Genome instability: a mechanistic view of its causes and consequences. Nat. Rev. Genet 9, 204–217.[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]
Blobel, G. (1985). Gene gating: a hypothesis. Proc. Nat. Acad. Sci. USA 82, 8527–8529.
Buratowski, S. (2005). Connections between mRNA 3' end processing and transcription termination. Curr. Opin. Cell Biol 17, 257–261.[CrossRef][Medline]
Cabal, G. G. et al. (2006). SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441, 770–773.[CrossRef][Medline]
Casolari, J. M., Brown, C. R., Komili, S., West, J., Hieronymus, H., and Silver, P. A. (2004). Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization. Cell 117, 427–439.[CrossRef][Medline]
Chaudhuri, J., Tian, M., Khuong, C., Chua, K., Pinaud, E., and Alt, F. W. (2003). Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730.[CrossRef][Medline]
Chavez, S., Beilharz, T., Rondon, A. G., Erdjument-Bromage, H., Tempst, P., Svejstrup, J. Q., Lithgow, T., and Aguilera, A. (2000). A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae. EMBO J 19, 5824–5834.[CrossRef][Medline]
Chekanova, J. A., Abruzzi, K. C., Rosbash, M., and Belostotsky, D. A. (2008). Sus1, Sac3, and Thp1 mediate post-transcriptional tethering of active genes to the nuclear rim as well as to non-nascent mRNP. RNA 14, 66–77.
Cole, C. N., and Scarcelli, J. J. (2006). Transport of messenger RNA from the nucleus to the cytoplasm. Curr. Opin. Cell Biol 18, 299–306.[CrossRef][Medline]
Dieppois, G., Iglesias, N., and Stutz, F. (2006). Cotranscriptional recruitment to the mRNA export receptor Mex67p contributes to nuclear pore anchoring of activated genes. Mol. Cell. Biol 26, 7858–7870.
Fan, H. Y., Merker, R. J., and Klein, H. L. (2001). High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability. Mol. Cell. Biol 21, 5459–5470.
Faria, A. M., Levay, A., Wang, Y., Kamphorst, A. O., Rosa, M. L., Nussenzveig, D. R., Balkan, W., Chook, Y. M., Levy, D. E., and Fontoura, B. M. (2006). The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions. Immunity 24, 295–304.[CrossRef][Medline]
Fischer, T., Rodriguez-Navarro, S., Pereira, G., Racz, A., Schiebel, E., and Hurt, E. (2004). Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery. Nat. Cell Biol 6, 840–848.[CrossRef][Medline]
Fischer, T., Strasser, K., Racz, A., Rodriguez-Navarro, S., Oppizzi, M., Ihrig, P., Lechner, J., and Hurt, E. (2002). The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J 21, 5843–5852.[CrossRef][Medline]
Gallardo, M., and Aguilera, A. (2001). A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination. Genetics 157, 79–89.
Gallardo, M., Luna, R., Erdjument-Bromage, H., Tempst, P., and Aguilera, A. (2003). Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism. J. Biol. Chem 278, 24225–24232.
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]
Garcia-Rubio, M., Chavez, S., Huertas, P., Tous, C., Jimeno, S., Luna, R., and Aguilera, A. (2008). Different physiological relevance of yeast THO/TREX subunits in gene expression and genome integrity. Mol. Genet. Genomics 279, 123–132.[CrossRef][Medline]
Gari, E., Piedrafita, L., Aldea, M., and Herrero, E. (1997). A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13, 837–848.[CrossRef][Medline]
Gómez-González, B., and Aguilera, A. (2007). Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex. Proc. Natl. Acad. Sci. USA 104, 8409–8414.
Gonzalez-Barrera, S., Garcia-Rubio, M., and Aguilera, A. (2002). Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae. Genetics 162, 603–614.
Govind, C. K., Zhang, F., Qiu, H., Hofmeyer, K., and Hinnebusch, A. G. (2007). Gcn5 promotes acetylation, eviction, and methylation of nucleosomes in transcribed coding regions. Mol. Cell 25, 31–42.[CrossRef][Medline]
Gwizdek, C., Iglesias, N., Rodriguez, M. S., Ossareh-Nazari, B., Hobeika, M., Divita, G., Stutz, F., and Dargemont, C. (2006). Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription. Proc. Natl. Acad. Sci. USA 103, 16376–16381.
Hecht, A., and Grunstein, M. (1999). Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction. Methods Enzymol 304, 399–414.[Medline]
Huertas, P., and Aguilera, A. (2003). Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol. Cell 12, 711–721.[CrossRef][Medline]
Huertas, P., Garcia-Rubio, M. L., Wellinger, R. E., Luna, R., and Aguilera, A. (2006). An hpr1 point mutation that impairs transcription and mRNP biogenesis without increasing recombination. Mol. Cell. Biol 26, 7451–7465.
Ivanovska, I., and Rose, M. D. (2001). Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication. Genetics 157, 503–518.
Jimeno, S., Rondon, A. G., Luna, R., and Aguilera, A. (2002). The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability. EMBO J 21, 3526–3535.[CrossRef][Medline]
Köhler, A., Pascual-Garcia, P., Llopis, A., Zapater, M., Posas, F., Hurt, E., and Rodriguez-Navarro, S. (2006). The mRNA export factor Sus1 is involved in Spt/Ada/Gcn5 acetyltransferase-mediated H2B deubiquitinylation through its interaction with Ubp8 and Sgf11. Mol. Biol. Cell 17, 4228–4236.
Köhler, A., Schneider, M., Cabal, G. G., Nehrbass, U., and Hurt, E. (2008). Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export. Nat. Cell Biol 10, 707–715.[CrossRef][Medline]
Kurshakova, M. M., Krasnov, A. N., Kopytova, D. V., Shidlovskii, Y. V., Nikolenko, J. V., Nabirochkina, E. N., Spehner, D., Schultz, P., Tora, L., and Georgieva, S. G. (2007). SAGA and a novel Drosophila export complex anchor efficient transcription and mRNA export to NPC. EMBO J 26, 4956–4965.[CrossRef][Medline]
Lei, E. P., and Silver, P. A. (2002). Intron status and 3'-end formation control cotranscriptional export of mRNA. Genes Dev 16, 2761–2766.
Lei, E. P., Stern, C. A., Fahrenkrog, B., Krebber, H., Moy, T. I., Aebi, U., and Silver, P. A. (2003). Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex. Mol. Biol. Cell 14, 836–847.
Li, X., and Manley, J. L. (2005). Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell 122, 365–378.[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]
Mason, P. B., and Struhl, K. (2005). Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo. Mol. Cell 17, 831–840.[CrossRef][Medline]
Mendjan, S. et al. (2006). Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila. Mol. Cell 21, 811–823.[CrossRef][Medline]
Mumberg, D., Muller, R., and Funk, M. (1994). Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22, 5767–5768.
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563.[CrossRef][Medline]
Okazaki, I. M., Kinoshita, K., Muramatsu, M., Yoshikawa, K., and Honjo, T. (2002). The AID enzyme induces class switch recombination in fibroblasts. Nature 416, 340–345.[CrossRef][Medline]
Piruat, J. I., and Aguilera, A. (1998). A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongation-associated recombination. EMBO J 17, 4859–4872.[CrossRef][Medline]
Prado, F., Piruat, J. I., and Aguilera, A. (1997). Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation. EMBO J 16, 2826–2835.[CrossRef][Medline]
Ragoczy, T., Bender, M. A., Telling, A., Byron, R., and Groudine, M. (2006). The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation. Genes Dev 20, 1447–1457.
Rehwinkel, J., Herold, A., Gari, K., Kocher, T., Rode, M., Ciccarelli, F. L., Wilm, M., and Izaurralde, E. (2004). Genome-wide analysis of mRNAs regulated by the THO complex in. Drosophila melanogaster. Nat. Struct. Mol. Biol 11, 558–566.[CrossRef][Medline]
Revy, P. et al. (2000). Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575.[CrossRef][Medline]
Rodriguez-Navarro, S., Fischer, T., Luo, M. J., Antunez, O., Brettschneider, S., Lechner, J., Perez-Ortin, J. E., Reed, R., and Hurt, E. (2004). Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75–86.[CrossRef][Medline]
Rondon, A. G., Garcia-Rubio, M., Gonzalez-Barrera, S., and Aguilera, A. (2003a). Molecular evidence for a positive role of Spt4 in transcription elongation. EMBO J 22, 612–620.[CrossRef][Medline]
Rondon, A. G., Jimeno, S., Garcia-Rubio, M., and Aguilera, A. (2003b). Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation. J. Biol. Chem 278, 39037–39043.
Santos-Rosa, H., and Aguilera, A. (1994). Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1 delta strains. Mol. Gen. Genet 245, 224–236.[Medline]
Strasser, K. et al. (2002). TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, 304–308.[CrossRef][Medline]
Sullivan, D. S., Biggins, S., and Rose, M. D. (1998). The yeast centrin, cdc31p, and the interacting protein kinase, Kic1p, are required for cell integrity. J. Cell Biol 143, 751–765.
Taddei, A., Van Houwe, G., Hediger, F., Kalck, V., Cubizolles, F., Schober, H., and Gasser, S. M. (2006). Nuclear pore association confers optimal expression levels for an inducible yeast gene. Nature 441, 774–778.[CrossRef][Medline]
Wyce, A., Xiao, T., Whelan, K. A., Kosman, C., Walter, W., Eick, D., Hughes, T. R., Krogan, N. J., Strahl, B. D., and Berger, S. L. (2007). H2B ubiquitylation acts as a barrier to Ctk1 nucleosomal recruitment prior to removal by Ubp8 within a SAGA-related complex. Mol. Cell 27, 275–288.[CrossRef][Medline]
Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E., and Lieber, M. R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol 4, 442–451.[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.
Zhao, Y. et al. (2008). A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing. Mol. Cell 29, 92–101.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  D. Roy and M. R. Lieber G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter Mol. Cell. Biol., June 1, 2009; 29(11): 3124 - 3133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Klockner, M. Schneider, S. Lutz, D. Jani, D. Kressler, M. Stewart, E. Hurt, and A. Kohler Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination J. Biol. Chem., May 1, 2009; 284(18): 12049 - 12056. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Faza, S. Kemmler, S. Jimeno, C. Gonzalez-Aguilera, A. Aguilera, E. Hurt, and V. G. Panse Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery J. Cell Biol., March 23, 2009; 184(6): 833 - 846. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
) and GL-ribm (
) are shown. All experiments were performed in 2% galactose to allow expression of the direct repeats. Lack (–RNH1) or overexpression (+RNH1) of RNase H1 was achieved with either p416-GAL1 or the multicopy plasmid pGAL-RNH1 carrying RNH1 under the GAL1 promoter, respectively. Recombination frequencies are the median value of six independent cultures. The average median value of 2–4 experiments and SD are shown.