MRN1 Implicates Chromatin Remodeling Complexes and Architectural Factors in mRNA Maturation

A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6ΔΔ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin architectural proteins Nhp6A/Nhp6B, accumulate intron-containing pre-mRNA at the restrictive temperature. In addition, we demonstrate that rsc8-ts16 nhp6ΔΔ cells contain low levels of U6 snRNA and U4/U6 di-snRNA that is further exacerbated after two hours growth at the restrictive temperature. This change in U6 snRNA and U4/U6 di-snRNA levels in rsc8-ts16 nhp6ΔΔ cells is indicative of splicing deficient conditions. We identify MRN1 (multi-copy suppressor of rsc nhp6ΔΔ) as a growth suppressor of rsc nhp6ΔΔ synthetic sickness. Mrn1 is an RNA binding protein that localizes both to the nucleus and cytoplasm. Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309Δ, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing.


Introduction
In eukaryotes, DNA is packaged into chromatin, which can inhibit the accessibility of DNA binding factors to their cognate sites in vivo. Thus, chromatin structural changes play a central role in controlling gene transcription as the formation of transcripts must contend with the repressive chromatin [1]. For active transcription to take place nucleosomes, the basic units of chromatin, need to be remodeled. ATP-dependent remodelers containing a catalytic subunit belonging to the Swi2/Snf2 family of ATPases, induce conformational changes in nucleosomes by altering histone-DNA interaction. In the Swi2/Snf2 family four different subclasses of remodelers are recognized: SWI/SNF, ISWI, CHD and INO80, that are all conserved from yeast to metazoans [2]. The yeast Saccharomyces cerevisiae contains the founding family member, SWI/SNF, and the highly related RSC (remodels the structure of chromatin) complex. RSC is abundant and holds fifteen-subunits with central roles in transcription [3,4], DNA repair [5] and chromosome segregation [6]. Moreover, a genome-wide location analysis indicated that RSC is recruited to both RNA polymerase II (RNAPII) and RNA polymerase III (RNAPIII) promoters [7] and recently it was shown that RSC regulates nucleosome positioning at RNAPII genes and nucleosome density at RNAPIII genes [8].
The S. cerevisiae chromatin architectural factors and histone modifiers Nhp6A/B are related to the high-mobility group 1 (HMG1) family of small, abundant chromatin proteins that lack sequence specificity of DNA binding, but bend DNA sharply and modulate gene expression [9]. Nhp6 is encoded by two genes, NHP6A and NHP6B, which are functionally redundant. Consequently, only the nhp6A nhp6B double deletion mutant (nhp6DD mutant) is temperature sensitive for growth [10]. Nhp6p is important for activation and repression of transcription of several RNAPII genes [11] and promote transcriptional elongation as part of the FACT complex [12]. Of significance in the context of this paper, Nhp6 is important for expression of the SNR6 gene, encoding the U6 snRNA transcribed by RNAPIII [13,14].
The human SWI/SNF subunit BAF57 contains a HMG box domain similar to the one present in Nhp6, which is not found in the yeast complex [15] and the Drosophila BRM component Bap111 is also a HMG-domain protein [16]. In yeast, NHP6 interacts genetically with both SWI/SNF and RSC [17], both RSC and Nhp6 have a repressive effect on the expression of CHA1 [3,11] and data from transcriptome analysis of swi/snf and nhp6DD mutants, partly overlap [11]. Furthermore, RSC components interact with Nhp6A in vitro and facilitate the loading of Nhp6A onto nucleosomes [17].
A connection between chromatin dynamics and mRNA processing has previously been suggested [18]. The SWI/SNF complex has been linked to alternative pre-mRNA splicing [19,20]. In higher eukaryotes pre-mRNA splicing is suggested to be a co-transcriptional event [21,22]. In yeast splicing mainly occurs post-transcriptionally, but initiation of spliceosome assembly and removal of introns from genes with long second exons are probably co-transcriptional events [23,24]. The spliceosome consists of 5 snRNPs (small nuclear ribonucleoprotein particles (U1, U2, U4, U5, U6)) as well as non-snRNP proteins [25,26]. Brg1, a subunit of the mammalian orthologue of the yeast SWI/ SNF complex interacts with hPrp4, a U5 snRNP-associated kinase [27]. Brm, also a subunit of the mammalian orthologue of the yeast RSC (SWI/SNF) complex, was found upon over-expression to favor inclusion of variant exons in the mRNA and to associate with both U1-and U5-snRNP as well as with coding regions of intron-containing genes [20]. Brm in insect cells was shown to be associated with nascent pre-mRNA's and to regulate the type of alternative transcripts produced [19]. Brm, Brg1 and additional SWI/SNF-related polypeptides associate with chicken supraspliceosomes [28]. Included in the supraspliceosome is the NineTeen Complex (NTC), which functions in spliceosome activation by specifying the interaction of U5 and U6 with pre-mRNA for their stable association with the spliceosome after U1 and U4 dissociation [29,30].
Here we take a genetic approach and discover an interplay between HMG proteins, chromatin remodeling factors and mRNA maturation. We show that rsc nhp6DD triple mutants accumulate pre-mRNA and demonstrate that rsc8-ts16 nhp6DD cells display low levels of the U4/U6 snRNA dimer and of total U6 snRNA. Thus, a link between chromatin remodelers, architectural factors and mRNA maturation is established.

Chromatin remodeling complexes and Nhp6 interact genetically
In Saccharomyces cerevisiae, the remodeling complex RSC and the architectural factors Nhp6 have a repressive effect on the chromatin structure at the CHA1 locus [3,11]. Release of both RSC-and Nhp6-dependent repression results in increased transcript levels of CHA1 mRNA, suggesting that RSC and Nhp6 co-operate in CHA1 repression [3,11]. To identify further relationships between RSC and Nhp6, we tested whether NHP6 genetically interacts with RSC or SWI/SNF and found that the swi2D nhp6DD and rsc8-ts21 nhp6DD triple mutants exhibited a synthetic sickness phenotype compared to their cognate single and double mutants ( Figure 1A and Figure 1B). The combination of rsc mutations rsc8-ts16, sfh1-1, sth1-3ts, rsc1D or rsc2D and swi/snf mutations swi3D, snf5D or snf6D with nhp6DD also resulted in reduced growth (Table 1). Thus, the architectural factor Nhp6 shares functionality with RSC and SWI/SNF.
Multi-copy growth suppression screen of rsc8-ts16 nhp6DD yields MRN1 Next we performed a suppression screen of the rsc8-ts16 nhp6DD synthetic sickness phenotype. Using a Yep24-based (2 mm) genomic library [31] we isolated YPL184c as a multi-copy suppressor ( Figure 1C) and named it MRN1 for multi-copy suppressor of rsc nhp6. Western blot analysis of Myc-tagged 2 mm-MRN1 confirmed increased levels of the Mrn1 protein ( Figure 1D). Multi-copy MRN1 was able to suppress the growth defect of all tested rsc nhp6 and swi/snf nhp6 triple mutants except rsc2D nhp6DD (Table 1). The latter result is likely indicative of the inability of the rsc2D mutant to maintain 2 mm plasmids [32].
The Mrn1 protein is predicted to be 612 amino acids long and to contain as many as five RNA Recognition Motifs (RRMs, Figure 1E). Four of these are arranged in pairs and within each pair the RRMs are separated by a short linker (,23 amino acids, Figure 1E). Present in all kingdoms of life, and most abundantly in eukaryotes, RRM domains are able to bind RNA and also DNA and protein(s) [33]. In addition to the predicted RRM domains, Mrn1 contains an N-terminal region rich in asparagine (91% between amino acids 6 and 28, Figure 1E), a region rich in glutamine (44% between amino acids 98 and 125, Figure 1E) and two regions rich in alanine (40-55% between amino acids 171-189 and amino acids 407-422 respectively, Figure 1E).

Cellular location of Mrn1
The cellular location of Mrn1-GFP expressed from its genomic location has been reported to be cytoplasmic [34] (http://yeastgfp. ucsf.edu/)). Using the same Mrn1-GFP tagged strain we also observed Mrn1 located primarily in the cytoplasm both at 25uC and 37uC ( Figure 2A). However, in these cells we estimated that approximately 5% of Mrn1 is nuclear (M. Lisby, personal communication). To unambiguously detect Mrn1 in the nucleus, we analyzed Mrn1 localization in the temperature-sensitive mex67-5 mRNA export deficient mutant [35]. In the mex67-5 genetic background we detected Mrn1-GFP accumulation in the nucleus at 37uC in approximately 95% of the cells (Figure 2A and Figure 2B). This demonstrates that Mrn1 is located both in the nucleus and the cytoplasm.

Genetic link between chromatin, MRN1 and mRNA processing
To substantiate the genetic link between MRN1 and chromatin remodeling complexes, we combined mrn1D with swi2D or nhp6DD, respectively. We found that the mrn1D swi2D combination resulted in synthetic sickness on plates containing 3% formamide ( Figure 3A), which is known to cause transcriptional stress, and that the mrn1D nhp6DD triple deletion was sick at 37uC ( Figure 3B). In contrast, the mrn1D snf5D or mrn1D rsc2D combinations did not reveal enhanced growth defects (data not shown).
The presence of RRM domains in Mrn1 could suggest a possible role of the protein in mRNP maturation. Interestingly, an ongoing Synthetic Genetic Array (SGA) screen with mrn1D as query linked MRN1 genetically to several splicing deficient mutants (SGA screen to be published elsewhere). Thus, combining mrn1D and the snt309D mutant deleted of the NineTeen Complex (NTC) subunit Snt309 resulted in synthetic sickness ( Figure 3C). Snt309 associates with the spliceosome simultaneously with or immediately after dissociation of U4 [36] and the snt309D mutant has a splicing defect that results in the accumulation of introncontaining pre-mRNA at the non-permissive temperature in vivo [36]. Also, we found that 2 mm-MRN1 interacted genetically with snt309D as 2 mm-MRN1 suppressed the ts-phenotype of the snt309D mutant strain ( Figure 3D). The synthetic sickness of mrn1D snt309D indicated that multi-copy MRN1 suppression of the ts-phenotype of snt309D mutant reflects relevance for endogenous MRN1 function. Constanzo et al. recently reported that mrn1D interacts genetically with prp4-1, prp22, and snt309D [37]. Interestingly, 2 mm-MRN1 also suppressed the ts-phenotype of prp22 ( Figure 3E), prp4-1 ( Figure 3F) and prp3-1 (data not shown). As 2 mm-MRN1 suppressed the swi/snf and the rsc nhp6DD triple mutants as well as snt309D mutant, we examined whether rsc, swi/ snf or nhp6DD interacted genetically with snt309D and found that both snf5D snt309D and rsc2D snt309D double mutants were synthetic sick ( Figure 3G). In agreement with this, Cairns and coworkers reported genetic interaction between snt309D and rsc7D [38]. We also revealed a synthetic lethal interaction between snt309D and nhp6DD by tetrad analysis. Out of 21 tetrads 12 did not contain the triple mutant and the remaining 9 each had 3 viable spores and the missing spore would have been the triple mutant (data not shown). To establish that snt309D and nhp6DD indeed are synthetic lethal, an snt309D nhp6DD heterozygous diploid was transformed with a URA3 containing plasmid expressing NHP6B. After dissection, genotype verification, and spot assay we found that snt309D nhp6DD spores were unable to grow on 5-FOA ( Figure 3H). However, Snt309p and Mrn1p are not functionally redundant as high-copy SNT309 did not suppress the growth defect of the rsc8-ts16 nhp6DD triple mutant (data not  Strains used in Table 1: SG759 (snf5D nhp6DD), SG469 (swi2D nhp6DD), SG476 (rsc2D nhp6DD), SG518 (rsc1D nhp6DD), SG657 (rsc8-ts16 nhp6DD), SG658 (rsc8-ts21 nhp6DD), SG659 (sfh1-1 nhp6DD), SG661 (sth1-3ts nhp6DD), SG662 (snf6D nhp6DD), SG742 (swi3D nhp6DD) and SG759 (swi2D nhp6DD). ND = Not determined. doi:10.1371/journal.pone.0044373.t001 shown). In addition, Mrn1 does not share genetic functionality with the RRM-containing Prp24 which mediates the re-annealing of the U4/U6 dimer [39] as high-copy MRN1 did not complement a prp24D mutant: dissection of 15 tetrads of a prp24D heterozygous diploid harboring 2 mm-MRN1 (pTK1395) all segregated 2:0 for viability. In conclusion, the genetic interactions shown in Figure 3 suggest a role for Mrn1, RSC, SWI/SNF and architectural factors in mRNA maturation.

rsc8-ts16 nhp6DD cells accumulate intron-containing pre-mRNA
To analyze if the rsc nhp6 mutation influences the amounts of intron-containing pre-mRNA in vivo we isolated total RNA from rsc8-ts16 nhp6DD and snt309D (as control) mutant strains grown at 25uC and after a two-hour incubation at 37uC. A Northern blot was probed for the intron-containing ECM33 pre-mRNA and the ECM33 39 exon mRNA ( Figure 4A -see Figure S1 for position of probes). Increased levels of pre-mRNA were observed in both mutants at 25uC and this increase was exacerbated after a two hour incubation at 37uC. We also observed that the amount of mature ECM33 mRNA at 37uC was strongly decreased in the two mutant strains as compared to wild type. The same analysis of the RPS11B pre-mRNA and mRNA also revealed that pre-mRNA levels in both mutants at 30uC were increased compared to the wild type and the levels of both pre-mRNAs were further increased after two hours incubation at 37uC. Again, the increase in RPS11B pre-mRNA were accompanied by a decrease in mature mRNA ( Figure 4B). To extend the analysis we measured the ratio of pre-mRNA to 39 exon mRNA by Reverse Transcriptase quantitative-PCR (RT-qPCR) of ECM33 transcripts. Indeed, the rsc8-ts16 nhp6DD triple mutant had increased in vivo pre-mRNA/39exon ratio of the ECM33 transcript already at 25uC and this effect was dramatically enhanced after two hours at 37uC ( Figure 4C). Although a small (1.5-fold) increase in overall transcription of ECM33 in the rec8-ts16 nhp6DD mutant was seen, the 55-fold relative increase in the unspliced ECM33 pre-mRNA at 37uC is 36 times higher than that of the relative increase in total ECM33 mRNA (compare Figure 4D and Figure 4E). Interestingly, the pre-mRNA accumulation phenotype at 37uC of rsc8-ts16 nhp6DD cells was partly suppressed by 2 mm-MRN1 ( Figure 4C). Similarly, RT-qPCR analysis of the three intron-containing genes ACT1, ASC1 and RPS11B in the rsc8-ts16 nhp6DD mutant revealed an accumulation of their pre-mRNA's at 25uC and exceedingly more so after incubation at 37uC for 2 hours ( Figure 4C). Again, the relative increase in total RNA levels at 37uC was lower (2-5-fold) than the relative increase in pre-mRNA levels (14-60-fold) (compare Figure 4D and Figure 4E). Furthermore, overexpressed Mrn1 modestly suppressed the accumulation of ACT1, ASC1, and RPS11B pre-mRNA at 37uC ( Figure 4C). Analyses of all four intron-containing transcripts in the rsc1D nhp6DD and snt309D mutants revealed a similar accumulation of pre-mRNA at 25uC and exceedingly more so after incubation at 37uC for two hours ( Figure S2). Importantly, accumulation of ECM33, ACT1, ASC1, RPS11B pre-mRNAs did not generally occur in single rsc mutants or in the double nhp6 deletion strain ( Figure S3). Thus, rsc nhp6 triple mutants exhibited an mRNA maturation deficiency, which was aggravated after a two hour incubation at 37uC. In addition to suppression of the temperature sensitivity of the rsc nhp6 triple and snt309D strains, Mrn1 over-expression also modestly suppressed the pre-mRNA accumulation exhibited by the mutants.
Reduced U4/U6 dimer snRNA levels in rsc nhp6DD cells In wild type cells U6 snRNP is in excess of U4 snRNP, but reduced levels of U6 is a common phenotype in strains with mutations in genes encoding U6, U4/U6, or tri-snRNP components including Prp3, Prp4, Prp19, Prp24, Prp38 and Lsm proteins [40,41,42,43,44,45,46]. Apparently, in these mutant strains the U4/U6 complex is destabilized. Specifically, in snt309D mutant cells the U4/U6 dimer is destabilized, resulting in accumulation of free U4 and decreased levels of total U6 and in failure of spliceosome recycling due to impaired U4/U6 biogenesis [47]. This is underscored as over-expressed U6 suppresses the tsphenotype of snt309D [47]. In addition, we found that 2 mm-SNR6 restored growth of the rsc8-ts16 nhp6DD triple mutant (data not shown). To determine the levels of the U4/U6 dimer, free U4 and total U6 in rsc8-ts16 nhp6DD cells total RNA was fractionated both on non-denaturing and denaturing polyacrylamide gels for Northern analysis with U4 and U6 specific probes, respectively. The rsc8-ts16 nhp6DD cells had decreased amounts of the U4/U6 dimer and accumulated free U4 at 25uC ( Figure 5A). Interestingly, the amount of U4/U6 dimer was further decreased after a twohour incubation at 37uC. In the mutant total U6 snRNA levels were also reduced after two hours at 37uC ( Figure 5B). To quantify the snRNA levels we performed five independent experiments and normalized the U4 and U6 data to the same blots re-probed for U1 snRNA. First, the rsc8-ts16 nhp6DD strain showed a more than 5-fold increase in free U4; second, at 25uC the mutant strain had a 2-fold decrease in the levels of U4/U6 and total U6; third, a twohour shift to 37uC resulted in a further, significant 1-2-fold reduction in U4/U6 and total U6 levels ( Figure 5C). We conclude that rsc8-ts16 nhp6DD cells contain low levels of U4/U6 dimer snRNA, of total U6 snRNA and accumulate free U4 snRNA at 25uC and that the low levels of U4/U6 dimer and of total U6 is further aggravated after a two-hour shift at 37uC. U4/U6 dimer and total U6 snRNA levels are unchanged after a two-hour transcriptional shutdown Both RSC and Nhp6 are involved in transcriptional regulation of RNAPIII transcribed genes and high-copy SNR6 suppresses the growth defect of nhp6DD double mutants [14,48]. Therefore, it was important to determine if the observed reduction in U4/U6 dimer and total U6 snRNA levels in the triple mutant was an effect of the rsc nhp6 mutations to reduce SNR6 transcription. In this case the drop in U4/U6 and total U6 snRNA content would just reflect SNR6 RNA turnover. We addressed this question by determining U4/U6 dimer, free U4 and total U6 stability in rsc8-ts16 nhp6DD cells after growth for two hours in the presence of thiolutin. The antifungal agent thiolutin efficiently inhibits all three yeast polymerases both in vivo and in vitro [49,50]. Total RNA was Figure 3. Genetic interactions linking MRN1 and chromatin mutants to pre-mRNA splicing. (A) mrn1D is synthetic sick with swi2D. Cells ten-fold serially diluted, spotted on SC plates or SC plates containing 3% formamide and incubated for four days at 30u. swi2D: SG418; mrn1D: SG520; wild type: SG632 and mrn1D swi2D: SG766. (B) mrn1D is synthetic sick with nhp6DD. Cells ten-fold serially diluted, spotted on SC plates and incubated for four days at the indicated temperatures. mrn1D: SG520; wild type: SG632; nhp6DD: SG727 and mrn1D nhp6DD: SG762. (C) mrn1D snt309D cells are synthetic sick. Cells ten-fold serially diluted, spotted on SC plates and grown at the indicated temperatures for four days. mrn1D: SG912; wild type: SG632; snt309D: SG648; mrn1D snt309D: SG920. (D) The temperature sensitivity of snt309D is suppressed by 2 mm-MRN1. Cells ten-fold serially diluted, spotted on SC-His plates and incubated for four days at the indicated temperatures. Wild type: SG632; snt309D: SG648; 2 mm-vector: pTK839; 2 mm-MRN1: pTK1395. (E) The temperature sensitivity of prp22 is suppressed by 2 mm-MRN1. Cells ten-fold serially diluted, spotted on SC-His plates and incubated for four days at the indicated temperatures. Wild type: SG682; prp22: SG840; 2 mm-vector: pTK839; 2 mm-MRN1: pTK1423. (F) The temperature sensitivity of prp4-1 is suppressed by 2 mm-MRN1. Cells ten-fold serially diluted, spotted on SC-Ura plates and incubated for four days at the indicated temperatures. prp4-1: SG845; 2 mm-vector: pTK51; 2 mm-MRN1: pTK1386. (G) snf5D and rsc2D genetically interacts with snt309D. Cells ten-fold serially diluted, spotted on SC plates and incubated for four days at the indicated temperatures. rsc2D: SG417; snf5D: SG420; wild type: SG632; snt309D: SG729; rsc2D snt309D: SG773 and snf5D snt309D: SG774. (H) snt309D is synthetic lethal with nhp6DD. Cells ten-fold serially diluted, spotted on SC-Ura plates or 5-FOA plates and incubated for four days at 30uC. Wild type: SG865; nhp6DD: SG867; snt309D: SG868; snt309D nhp6DD: SG869; 2 mm-NHP6B-URA3: pTK1382. Colony rows compared in the same panel derives from one plate. doi:10.1371/journal.pone.0044373.g003 Figure 4. rsc8-ts16 nhp6DD cells accumulate unspliced transcripts. (A) Northern blot analysis was done with total RNA isolated from logarithmically SC-His growing cells at 25uC or after a two hour shift at 37uC. Total RNA was electrophoresed in a 0.25 M formaldehyde agarose gel, blotted and hybridized with specific 32 P-labeled probes. The probe was either intron-specific or 39 exon-specific, respectively, for the ECM33 RNA (see Figure S1). Ethidium bromide staining of the 18S and 25S rRNA is shown as a loading control. (B) Northern blot analysis was done with total RNA isolated from logarithmically SC-His growing cells at 30uC or after a two hour shift at 37uC. The probe was specific for both the RPS11B pre-mRNAand for the RPS11B mRNA (see Figure S1). Ethidium bromide staining of the 18S and 25S rRNA is shown as a loading control. isolated from cells treated with thiolutin for two hours and the levels of U4/U6, free U4, and total U6 was determined by Northern blotting. The amount of U4/U6 dimer, free U4 and total U6 was unchanged after two hours of incubation with thiolutin both in the wild type and more importantly, also in the rsc8-ts16 nhp6DD strain ( Figure 6A and Figure 6B). Quantification of four independent experiments confirmed this result ( Figure 6C). In contrast, as expected the levels of three tested mRNA's were drastically reduced under the same growth conditions ( Figure 6D). Thus, efficiently shutting down RNA polymerase III transcription by the polymerase inhibitor thiolutin for two hours did not influence the levels of U4/U6 dimer or total U6 snRNA neither in the wild type nor in the rsc8-ts16 nhp6DD strain indicating that the decrease in snRNA levels is not due to impaired transcription of the SNR6 gene, but only observed in cells with specific splicingdeficient conditions.

Discussion
In this study we have utilized a genetic approach to study the functional interplay between the chromatin remodeling complexes RSC or SWI/SNF and the architectural factors Nhp6. We found that rscor swi/snf mutations in combination with nhp6 double deletion results in synthetic sickness. Interestingly, we found that rsc nhp6 triple mutants accumulate pre-mRNA, strongly suggesting a defect in pre-mRNA maturation. The defect in pre-mRNA maturation is underscored as rsc8-ts16 nhp6DD cells contained low levels of U4/U6 dimer and total U6 snRNA as well as high amounts of free U4 snRNA. Further, incubation at 37uC for two hours dramatically enhanced the accumulation of pre-mRNA in rsc nhp6DD cells. This is substantiated as rsc8-ts16 nhp6DD cells contained significantly reduced amounts of U4/U6 dimer and total U6 after two hours incubation at 37uC. The reduction in U4/ U6 dimer and total U6 was not due to deficient SNR6 transcription as a two-hour shutdown of SNR6 transcription induced by thiolutin did not result in reduced amounts of U4/U6 dimer or total U6 indicating that these RNAs are very stable. In agreement with this result, U6 snRNA have previously been reported to be very stable unless in a splicing deficient mutant background [40]. For example, temperature inactivation of the known U6 (or U4/ U6) snRNP associated factors Prp3, Prp4, Prp6, Prp24 or the NTC or Prp38 splicing factors lead to a decrease in U6 snRNA levels [40,43,44,45,46,47]. Apparently, in these mutant strains the U4/U6 complex is destabilized, perhaps exposing the U6 snRNA to intracellular nuclease attack. Furthermore, Moenne et al. [51] observed only a slight decrease in total U6 snRNA level after a five hour inactivation of a temperature-sensitive RNAPIII mutant. Accordingly, a two-hour shift to 37uC reduces U4/U6 dimer and total U6 snRNA levels in rsc8-ts16 nhp6DD cells as a consequence of their mRNA processing defect and not as a consequence of deficient transcription of SNR6. We did not see a general accumulation of unspliced mRNA for the tested transcripts after a two hour incubation at 37uC in the rsc1D or rsc8-ts16 single mutants, or in the nhp6DD double mutant. However, the rsc and swi/snf single mutants, and the nhp6DD double mutant might harbor potential splicing defects. In support of this notion we observed that combining rsc2D or snf5D with the NTC splicing mutant snt309D resulted in synthetic sickness and that a snt309D nhp6DD triple mutant is synthetic lethal. In conclusion, the Figure 5. U4/U6 dimer, free U4 and total U6 snRNA levels in rsc8-ts16 nhp6DD cells. Total RNA prepared from logarithmically SC-His growing cells at 25uC or after a two-hour shift at 37uC. (A) RNA was fractionated on a non-denaturing 6% polyacrylamide gel, blotted and hybridized with a U4 specific probe. After analysis the membrane was stripped and re-probed with a U1 specific probe. (B) RNA was fractionated on a denaturing 6% polyacrylamide gel, blotted and hybridized with a U6 specific probe. After analysis the membrane was stripped and re-probed with a U1 specific probe. (C) Quantification of U4/U6 dimer, free U4 and total U6 snRNA amounts relative to U1 snRNA based on quantification of Storm Images from at least five individual experiments. Wild type: SG632; rsc8-ts16 nhp6DD: SG657. doi:10.1371/journal.pone.0044373.g005 combination of mutations in RSC and chromatin architectural factors results in a severe defect in pre-mRNA maturation.
We identified the RNA-binding protein Mrn1 as a multi-copy suppressor of the synthetic sickness of the rsc8-ts16 nhp6DD mutant. Mrn1 is predicted to contain five RRM domains present in many RNA-binding proteins taking part in all mRNA co-and posttranscriptional processing events [33]. Recently, Hogan et al. [52] reported that Mrn1 is an RNA-binding protein and interacts with 378 RNAs including ECM33 and ACT1. Our genetic interaction analysis of MRN1 and 2 mm-MRN1 lead to the discovery that rsc nhp6DD cells display a splicing deficient phenotype as discussed above. In addition, the genetic analysis might also suggest a role of Mrn1 in pre-mRNA maturation. Over-expression of Mrn1 suppressed the ts-phenotype of the rsc nhp6 triple mutants as well as that of the NTC subunit mutant snt309D. Combining mrn1D with snt309D resulted in synthetic sickness, but Mrn1 and Snt309 are not functionally redundant as only 2 mm-MRN1, and not 2 mm-SNT309 suppresses the rsc8-ts16 nhp6DD triple mutant phenotype. Additionally, Mrn1 does not share genetic functionality with the RRM-containing Prp24. Prp24 mediates the re-annealing of the U4/U6 dimer [39], but 2 mm-PRP24 does not suppress the tsphenotype of snt309D cells [47] and 2 mm-MRN1 does not suppress lethality of a prp24D mutant. However, the suppression is specific, at least to a certain degree, as over-expression of either Pub1 or human PTB, two RNA binding proteins with two pairs of RRM domains arranged as those of Mrn1, does not suppress the growth defect of the rsc8-ts16 nhp6DD triple mutant (J. Christiansen and S. Holmberg, unpublished data). Work in progress in our lab is trying to identify a specific event in the mRNA processing pathway where Mrn1 functions.
The observed pre-mRNA accumulation in the rsc nhp6 triple mutants can be explained in several ways. The lack of RSC/Nhp6 activity concomitantly might influence transcription of splicing factor-encoding genes leading to the observed pre-mRNA accumulation and U4/U6 destabilization. It is also possible that the primary splicing block imposed by the rsc8-ts16 nhp6DD mutant results from the failure of splicing complexes to assembly or function properly. Thus, RSC and Nhp6 might be required for generating the correct chromatin state required for proper spliceosome assembly thereby affecting mRNA processing. Recent studies document connections between chromatin and splicing. The mammalian orthologue of the RSC complex, hSWI/SNF subunit Brm, was found to associate with several components of the spliceosome as a regulator of alternative splicing in several mammalian cell types [20]. Likewise, Brm and several hSWI/SNF subunits were shown to associate with chicken supraspliceosomes [28]. In yeast only very few genes contain more than one intron, and although it has been reported that most splicing is posttranscriptionally, recruitment of U1 is a co-transcriptional event at probably all genes [24]. One possibility is that rsc nhp6 and swi/snf nhp6 cells are deficient in the process of co-transcriptional recruitment of the pre-spliceosome. Batsché et al. [20] showed that Brm interacts in vivo with both U1 and U5 snRNPs and suggested that hSWI/SNF is involved in recruitment of the splicing machinery. Tyagi et al. [19] recently showed that Brm interacts directly with nascent pre-mRNP's and suggest that Brm post-transcriptionally regulates the type of alternative transcript produced. Whether RSC, SWI/SNF and/or Nhp6 factors can be loaded onto pre-mRNA in yeast remains to be elucidated. Figure 6. U4/U6 dimer, free U4 and total U6 snRNA levels remain stable after two hours of transcriptional shutdown. Total RNA prepared from logarithmically SC-His growing cells at 25uC or after two hours growth in the presence of 5 mg/ml Thiolutin at 25uC. (A) RNA was fractionated on a non-denaturing 6% polyacrylamide gel, blotted and hybridized with a U4 specific probe. After analysis the membrane was stripped and re-probed with a U1 specific probe. (B) RNA was fractionated on a denaturing 6% polyacrylamide gel, blotted and hybridized with a U6 specific probe. After analysis the membrane was stripped and re-probed with a U1 specific probe. (C) Quantification of U4/U6 dimer, free U4 and Total U6 snRNA amounts relative to U1 snRNA based on quantification of Storm Images from at least four individual experiments. (D) Total RNA was isolated from logarithmically SC-His growing rsc8-ts16 nhp6DD cells at 25uC and electrophoresed in a 0.25 M formaldehyde agarose gel or in a denaturing 6% polyacrylamide gel, blotted and hybridized with gene-specific 32 P-labeled probes. rsc8-ts16 nhp6DD : SG657. doi:10.1371/journal.pone.0044373.g006

Media, strains and genetic methods
Yeast extract-peptone-dextrose (YPD) medium, synthetic minimal (SD) medium, synthetic complete (SC) and SC lacking specific amino acids were prepared as described previously [53]. Standard yeast methods were used for dissection, sporulation, mating and replica plating. Lithium acetate transformation was employed [54]. Yeast strains are listed in Table 2, plasmids in Table 3, and oligonucleotides in Table 4.

Multi-copy suppressor screen
Strain SG657 (rsc8-ts16 nhp6DD) was transformed with a Yep24 based yeast genomic library [31]. Colonies able to grow at the non-permissive temperature (34uC) were selected. In total ,2610 7 transformants were screened. Plasmids from ,40 colonies were rescued in Escherichia coli and 17 different plasmids were identified as suppressors. Sixteen contained either NHP6A or NHP6B. One plasmid, pTK1385, contained the genomic sequence from 190959 to 198486 of chromosome XVI. Subcloning revealed that plasmid pTK1386 (pTK1385 digested with NheI and SacI, blunt ended and re-ligated) containing the genomic sequence from 194878 to 198486 of chromosome XVI was a suppressor of rsc8-ts16 nhp6DD synthetic sickness. pTK1386 contained YPL184c as the only complete ORF. pTK1386 was digested with SnaBI and EcoRI and the 2357 bp fragment containing YPL184c, from 198277 to 195919 of the genomic sequence, was cloned into SmaI and EcoRI digested pRS423 (pTK839) resulting in plasmid pTK1395. pTK1395 was transformed into strain SG657 and was able to suppress its growth defect, and accordingly, we concluded that 2 mm-YPL184c is a suppressor of the synthetic sickness of the rsc8-ts16 nhp6DD triple mutant.

Construction of MRN1-MYC
The endogenous MRN1-MYC was constructed by inserting a Myc-Tag C-terminally on the MRN1 gene by transformation and homologous recombination in yeast strain TG694 with two PCR fragments using KANMX6 as the selection marker. DNA was amplified with oligonucleotides MYCa, sfh1d, sfh1e and MYCab using pTK1259 as the template. The manipulated region was subsequently sequenced to verify correct insertion.
2 mm-MRN1 was Myc-tagged by inserting a Myc-tag Cterminally in the MRN1 gene using SacI-linearized plasmid pTK1395 and transformation and homologous recombination in yeast with a Myc-Tag containing PCR fragment. DNA was amplified with oligonucleotides MYCc and MYCab using pTK1259 as the template. Plasmid pTK1423 was rescued in Escherichia coli from His + yeast transformants and sequenced to verify correct insertion. 2 mm-MRN1-MYC suppresses the synthetic sickness of the rsc8-ts16 nhp6DD triple mutant (data not shown).

Immuno blotting
Whole-cell extracts were prepared from 50 ml of cells growing exponentially in SC-His medium. Cells were collected and washed twice in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 5 mM MgCl 2 , 0.5 mM dithiothreitiol (DTT), 0.25% NP-40) supplemented with Complete protease inhibitor cocktail (Roche). Cells were re-suspended in 400 ml lysis buffer and then lysed with glass beads (SIGMA) in a bead mill for 3620 sec at 4uC. The cell debris was eliminated by centrifugation twice at 4uC (106g, 5 min and 25 min, respectively). Protein concentration was measured with Bio-Rad Dc Protein Assay. Proteins were separated by SDS-PAGE and transferred to a mixed cellulose ester membrane and immuno-blotted with primary anti-Myc antibody (C3956-Sigma) or anti-HA antibody (12CA5 Roche). Proteins were visualized with anti-Rabbit or anti-Mouse Immuno-globulins/HRP (DAKO) and ECL Plus (GE Healthcare) with Hyperfilm ECL (GE Healthcare).

Northern protocol
RNA was electrophoresed in a 0.25 M formaldehyde agarose gel, transferred to a Hybond-NX (GE Healthcare) membrane by blotting overnight. RNA was cross-linked to the membrane in a Stratalinker (1200 mJ/cm 2 ). Radioactively ( 32 P) random primed labeled probes were produced with Prime-ItH II Random Primer Labeling Kit (Stratagene) and purified with ProbeQuant G-25 Micro Colums (Amersham), utilizing gel purified PCR product as the template. The templates were produced with specific primers (Table 4) utilizing genomic yeast DNA as the template. Membranes were hybridized over night in a Hybaid oven at 42uC with Ultrahyb hybridization buffer (Ambion) and the membranes were washed as recommended by the manufacture. Hybridized probe were visualized and quantified using a Storm 840 Phosphorimager (Molecular Dynamics) and also visualized with Kodak BioMax MS Film when needed.
Measurement of pre-mRNA accumulation by RT-qPCR RNA was purified from exponentially growing cells with RNeasy Mini Kit (Qiagen). QuantiTect SYBR Green RT-PCR Kit (Qiagen) supplied with Fluorescein Calibration Dye (10 nM)  (BIO-RAD) was used for the RT-qPCR amplifications done with the iCycler iQ (BIO-RAD). Data was analyzed with the iCycler iQ software (BIO-RAD). Standard deviations were calculated as suggested by Simon [56]. The sequence of used oligonucleotides is shown in Table 4.

U4/U6 Assay
To visualize U4/U6 dimer, U4 Free and total U6 exponentially growing cells were harvested and resuspended in 250 ml of RNA extraction buffer (100 mM LiCl, 1 mM EDTA, 100 mM Tris-Cl (pH 7.5), 0.2% SDS) and transferred to a tube containing 250 ml glass beads and 250 ml Phenol-chloroform-isoamyl alchohol (25:5:0.2). Then the cells were lysed in a bead mill for 3615 sec at 4uC. For non-denaturing gels the aqueous phase containing the RNA was mixed with one-third volume of loading dye (50% glycerol, 0.02% bromophenol blue) and loaded on a 6% nondenaturing polyacrylamide (29:1) Tris-borate-EDTA gel containing 5% glycerol with 0.5 TBE as running buffer. The gel was run over night at 80 V at 4u. The gel was then soaked twice in 20 mM NaPO 4 (pH 6.5), 8.3 M urea, 0.1% SDS at 37u, for 45 min and once in 20 mM NaPO 4 (PH 6.5) at 4u for 1 hour. RNA was electrotransferred to a nylon membrane (Hybond-NX (GE Healthcare)) followed by UV cross-linking to the membrane in a Stratalinker (1200 mJ/cm 2 ). For denaturing gels the aqueous phase was mixed with one volume of 26RNA loading dye (Fermentas), denatured (70u for 10 min, on ice for 3 min) and loaded on a 6% denaturing polyacrylamide (29:1) Tris-borate-EDTA gel containing 5% glycerol with 0.56TBE as running buffer. Then the same protocol was used as for non-denaturing gels except the gel was only washed once in 20 mM NaPO 4 (pH 6.5), 8.3 M urea, 0.1% SDS. Radioactively ( 32 P) random primed labeled probes were produced with Prime-ItH II Random Primer Labeling Kit (Stratagene) and purified with ProbeQuant G-25 Micro Colums (Amersham) with single stranded oligonucleotides Snr14 or Snr6a as templates ( Table 4). The template for the U1 probe was generated by PCR using genomic yeast DNA and primers Snr19a and Snr19b (Table 4). Hybridization was at 42u with Rapid-Hyb buffer (GE Healthcare) in a Hybaid oven over night followed by 265 min washes in 66SSC, 0.2% SDS and one 15 min wash in 26SSC, 0.2% SDS at 42u. Hybridized probe were visualized and quantified using a Storm 840 Phosphorimager (Molecular Dynamics) and also visualized with Kodak BioMax MS Film when needed. This protocol was modified from Lygerou et al. [57]. Figure S1 Schematic representation of RPS11B, ASC1, ACT1, and ECM33 probes and qPCR primers. The relative position of the DNA fragments used as RPS11B and ECM33 Northern probes as well as the relative position of the primers used for the qPCR analyses are depicted. (TIF) Figure S2 rsc1D nhp6DD and snt309D cells accumulate unspliced transcripts. Total RNA isolated from logarithmically SC-His growing cells at 25uC or after a 2 hour shift at 37uC amplified by RT-qPCR with ECM33-, ACT1-, ASC1or RPS11B-specific primers. The ratio intron-39exon junction RT-PCR-amplificate/ 39exon RT-PCR-amplificate. The ratio in wild type cells at 25uC was arbitrarily set to 1. ND: Not determined. Wild type: SG632; rsc1D nhp6DD: SG518; snt309D: SG648. (TIF) Figure S3 rsc1D and rsc8-ts16 or nhp6DD mutants do not generally accumulate unspliced mRNA at 37uC. Total RNA isolated from logarithmically SC-His growing cells at 25uC or after a 2 hour shift at 37uC amplified by RT-qPCR with ECM33-, ACT1-, ASC1or RPS11B-specific primers. The ratio intron-39exon junction RT-PCR-amplificate/39exon RT-PCR-amplificate. The ratio in wild type cells at 25uC was arbitrarily set to 1. Wild type: SG632; rsc1D: SG416, rsc8-ts16: SG360 and nhp6DD: SG306. (TIF)