An hpr1 point mutation that impairs transcription and mRNP biogenesis without increasing recombination

MOLECULAR AND CELLULAR BIOLOGY, Oct. 2006, p. 7451–7465 Vol. 26, No. 20 0270-7306/06/$08.00⫹0 doi:10.1128/MCB.00684-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved. An hpr1 Point Mutation That Impairs Transcription and mRNP Biogenesis without Increasing Recombination 䌤 Pablo Huertas, Marı´a L. Garcı´a-Rubio, Ralf E. Wellinger, Rosa Luna, and Andre´s Aguilera* Departamento de Gene´tica, Facultad de Biologı´a, Universidad de Sevilla, Avd. Reina Mercedes 6, 41012 Sevilla, Spain Received 20 April 2006/Returned for modification 13 June 2006/Accepted 4 August 2006 THO/TREX, a conserved eukaryotic protein complex, is a key player at the interface between transcription and mRNP metabolism. The lack of a functional THO complex impairs transcription, leads to transcriptiondependent hyperrecombination, causes mRNA export defects and fast mRNA decay, and retards replication fork progression in a transcription-dependent manner. To get more insight into the interconnection between mRNP biogenesis and genomic instability, we searched for HPR1 mutations that differentially affect gene expression and recombination. We isolated mutants that were barely affected in gene expression but exhibited a hyperrecombination phenotype. In addition, we isolated a mutant, hpr1-101, with a strong defect in transcription, as observed for lacZ, and a general defect in mRNA export that did not display a relevant hyperrecombination phenotype. In THO single-null mutants, but not in the hpr1 point mutants studied, THO and its subunits were unstable. Interestingly, in contrast to hyperrecombinant null mutants, hpr1-101 did not cause retardation of replication fork progression. Transcription and mRNP biogenesis can therefore be impaired by THO/TREX dysfunction without increasing recombination, suggesting that it is possible to separate the mechanism(s) responsible for mRNA biogenesis defects from the further step of triggering transcriptiondependent recombination. Gene expression requires the correct coupling of transcription to mRNA processing and export (7, 17, 30, 31). The final outcome of transcription is a ribonucleoprotein particle (mRNP) consisting of an mRNA molecule bound to proteins, which later function in export, mRNA surveillance, and translation. Transcription and assembly of the mRNP are tightly coordinated with the export of mRNA (42). A number of proteins have been identified that are important for the correct assembly of export-competent mRNP particles, including the THO complex, composed of Tho2, Hpr1, Mft1, and Thp2, as identified in Saccharomyces cerevisiae (9). THO has also been purified from humans and Drosophila, in which Hpr1 and Tho2 homologues are present (32, 40). In yeast and humans, it has been shown that THO is part of the TREX complex, which includes the Sub2/UAP56 and Yra1/Aly mRNA export factors (40). Functional connections between THO and the Sub2 and Yra1 export factors have been shown by synthetic lethal phenotypes of double mutants and by multicopy suppression (11, 19, 40). Null mutants of the THO complex are impaired in transcription elongation (8, 23, 36). Additionally, these mutants exhibit a plethora of phenotypes, including transcription-dependent hyperrecombination (2), nuclear mRNA retention (40), and fast mRNA degradation (22, 46), as well as synthetic lethality in combination with mutations affecting transcription elongation such as spt4 (35) and with RNA export mutations such as mex67-5and yra1-1(19, 40). Altogether, these data suggest that THO plays a crucial role in mRNP metabolism at the interface between transcription and mRNP export. Mutants with changes affecting the Sub2-Yra1 component of TREX, the Mex67-Mtr2 mRNA export factor, the Nab2 hnRNP, or the Thp1-Sac3-Sus1 RNA export complex exhibit transcription and genetic instability phenotypes similar to those of THO mutants (12, 14, 19). The THO complex is exclusively recruited to transcribed chromatin (20, 40, 46). Recruitment of the Sub2 and Yra1 components of the THO/TREX complex to transcribed DNA is partially dependent on THO (46). In contrast to THO, binding of Sub2 to transcribed chromatin is RNA dependent (1). The relevance of THO in the early steps of mRNP biogenesis is supported by the observation that transcription impairment in hpr1⌬mutants is suppressed if the nascent mRNA is selfcleaved by an artificially engineered hammerhead ribozyme. In these mutants, the nascent mRNA can form RNA-DNA hybrids (R loops) linked to both impairment of transcription and transcription-associated recombination (18). The observation that depletion of the vertebrate ASF/SF2 splicing factor in chicken DT40 cells and human HeLa cells also leads to genomic instability linked to R loop formation (21) indicates that a number of mRNA processing enzymes may have a role in preventing RNA-dependent structures that trigger genome instability. Our current view is that the THO complex participates in cotranscriptional assembly of export-competent mRNP during transcription elongation and contributes to the prevention of the RNA from forming structures that hamper transcription and could compromise genome stability (3). Despite the link between impaired transcription and hyperrecombination in THO mutants, it is not clear whether impaired gene expression by itself is sufficient to induce hyperrecombination or whether hyperrecombination is possible in the absence of impaired gene expression. Thus, we searched for THO mutants compromised in gene expression that did not affect genetic integrity and vice versa. Hyperrecombination * Corresponding author. Mailing address: CABIMER, Av. Americo Vespucio s/n, 41092 Seville, Spain. Phone: 34 954 468372. Fax: 34 954 461664. E-mail: [email protected]. 䌤 Published ahead of print on 14 August 2006. 7451 on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from mutations that did not have a significant effect on transcription were not found. Instead, we isolated a new allele, hpr1-101 (L586P), which causes a strong decrease in transcript accumulation but does not show a relevant hyperrecombination phenotype. The in vivo stability of THO and its subunits was strongly reduced in THO-null mutants such as hpr1⌬but not in hpr1 point mutants. The absence of associated hyperrecombination seems to be due to the fact that, in contrast to hpr1⌬, transcription impairment in hpr1-101 does not hamper replication fork progression. Our results indicate that THO dysfunction can cause transcription defects that are not linked to impairment of replication fork progression, opening the possibility that THO links optimal mRNP biogenesis with transcription elongation, apart from its effect on genomic integrity. MATERIALS AND METHODS Strains and plasmids. The strains used in this study are listed in Table 1. All of the strains are isogenic to W303-1A, except LAU3-10A and LAU3-10D, which are congenic segregants from a SChY58A ⫻AYW3-1B cross. Strains WH101-1A and WH103-1A, isogenic to W303-1A but harboring the hpr1-101 and hpr1-103 alleles in the HPR1 chromosomal locus, were obtained by gene replacement in SChY58a (hpr1⌬::KAN). Plasmids pRS313, pRS316 (38), YCp70, YCpA13 (4), pRS314GLlacZ (15), YEp351-SUB2, pCM184-LAUR (19), pRS316-GAL1lacZ, pSCh202, pSCh204, pSCh206 (8), pPHO5-Rib ⫹ -lacZ (Rib ⫹ ), pPHO5-rib m -lacZ (rib m ), pGL-Rib ⫹ , pGL-rib m (18), and pRWY005 (43) have been described previously. Plasmid pRS313-GZ was constructed by cloning the GAL1pr::lacZ fusion from pRS316-GAL1lacZ into pRS313 in vivo as previously described (29). pRS316-HPR1 was obtained by cloning the 6.55-kb HindIII HPR1 fragment from YCpA13 into the HindIII site of pRS316. Plasmids YCphpr1-101 to YCphpr1-104 were obtained by in vivo cloning of linear mutagenized HPR1 PCR fragments into YCp70 in yeast strain LAU3-10A as described below. New hpr1 alleles obtained by random mutagenesis. In vitro mutagenesis of HPR1 was performed according to reference 27, with some modifications. PCR amplification of HPR1 was done under suboptimal conditions with 3.25 mM MgCl 2 , 0.25 mM MnCl 2 , 0.2 mM dTTP, 0.2 mM dGTP, 0.2 mM dCTP, 0.04 mM dATP, and 1.75 U of Expand High Fidelity Taq polymerase (Roche). YCpA13 (1 ␮g) was used as the template, and the oligonucleotides 5⬘GCCACGTTTGTT TACTCG 3⬘and 5⬘CTATCATCCAACGCTTCC 3⬘were used as primers. The LAU3-10A strain (hpr1⌬), previously transformed with pRS313-GZ, was cotransformed with the purified, PCR-mutagenized HPR1 mixture and the linear BamHI-XbaI fragment of YCpA13, which corresponds to YCp70 carrying 200 bp of HPR1 flanking sequences on each side. In vivo recombination between the PCR-induced hpr1 alleles and the linear vector led to formation of the YCphpr1 plasmids in the cells. Transformants thus obtained were plated on synthetic complete (SC) medium without histidine and leucine containing limited amounts of adenine (1.5 mg/liter) for visualization of red sectors resulting from recombination of the chromosomal leu2-k::ADE2-URA3::leu2-krepeat system. Colonies were then replicated onto SC without histidine and leucine containing 2% galactose to allow expression of the GAL1pr::lacZ fusion. On the following day, a solution containing 0.25 M phosphate buffer (pH 7.5), 0.1% sodium dodecyl sulfate (SDS), 400 ␮g/ml 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X- Gal), and 0.5% agarose was added and ␤-galactosidase activity was scored by blue color formation after2hat30°C. Colonies transformed with either YCpA13 or YCp70 were used as positive (wild type) or negative (hpr1⌬) controls, respectively. Purification of the THO complex and analysis of its subunits. The THO complex was purified by using the C-terminally tandem affinity purification (TAP) epitope-tagged Tho2 protein and following standard procedures (33) and using specifications previously reported (19). Mft1 was detected by Western analysis of purified THO complexes previously separated by 10% SDS-poly- acrylamide gel electrophoresis (PAGE) and transferred to nylon membranes. After treatment with phosphate-buffered saline containing 0.1% Tween 20 and 5% milk, proteins were detected with anti-Mft1 antibody and peroxidase-conju- gated goat anti-rabbit immunoglobulin G (IgG). Blots were washed with phosphate-buffered saline–0.1% Tween 20 and developed by enhanced chemiluminescence reactions (Amersham). The Tho2-TAP epitope fusion was detected in whole cell extracts with peroxidase antiperoxidase (PAP; Sigma) antibody by following the manufacturer’s instructions as described previously (19). 2D gel analysis of replication intermediates. Cells were grown overnight to an optical density at 600 nm of 0.2 to 0.3 in SC medium containing 1% glycerollactate and 1% raffinose as a carbon source. After bringing the culture to 2% galactose, cells were grown for another 3.5 to 4 h prior to DNA isolation. DNA of 100 ml of cells was isolated with cetyltrimethylammonium bromide (44), dissolved in a final volume of 100 ␮l of Tris-EDTA, and stored at 4°C. Prior to analysis, 50 ␮l of DNA was double digested with SmaI-SacI. After digestion, DNA was precipitated and subjected to two-dimensional (2D) gel electrophoresis as described previously (6), with the following modifications. DNA was separated for 20 h in the first dimension on a 0.5% agarose gel at 1 V/cm and for 14 h in the second dimension on a 1.2% agarose gel at 4 V/cm and 4°C in the presence of 0.3 ␮g/ml ethidium bromide. After alkaline transfer of the DNA onto a Hybond-N ⫹ membrane, the membrane was hybridized against a 32 P-labeled 1.2-kb fragment obtained by PCR covering ARS1 and part of the 3⬘-lacZ gene. Membranes were exposed on a Fuji 3600 Phosphorimaging system and analyzed with the imaging software supplied. ChIP. Recruitment of Sub2, Tho2, and RNAPII to chromatin was determined by chromatin immunoprecipitation (ChIP) analyses on the endogenous PMA1 gene in yeast strains containing a 5⬘-end TAP epitope-tagged version of either the SUB2 or the THO2 gene inserted at its chromosomal locus, as previously described (16). Briefly, immunoprecipitations were performed with Ig-Sepharose for TAP epitope-tagged proteins or with anti-Rpb1-CTD monoclonal antibody TABLE 1. Yeast strains used in this study Strain Genotype Source or reference W303-1A MATaade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1R. Rothstein AYW3-1B MAT␣ade2 can1-100 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k13 U768-4C W303-1A hpr1⌬::HIS3 R. Rothstein SChY58a W303-1A hpr1⌬::KAN S. Cha´vez WH101-1A W303-1A hpr1-101 This study WH103-1A W303-1A hpr1-103 This study BSU-S2T-6D MATaade2-1 leu2 hpr1⌬::HIS3 SUB2-TAP 19a LAU3-10A MATaade2-1 his3-11,15 trp1-1 ura3-1This study leu2-k::ADE2-URA3::leu2-k hpr1⌬::KAN LAU3-10D MAT␣ade2-1 his3-11,15 trp1-1 ura3-1 leu2-k::ADE2-URA3::leu2-kThis study WMC1-1A W303-1A mex67-519 WMH1 hpr1⌬::KAN mex67-5isogenic to W303-1A and transformed with pRS316-HPR1 This study WWT2T W303-1A THO2-TAP 19a WTT3-4A W303-1A THO2-TAP hpr1-101 This study WTT3-4C W303-1A THO2-TAP hpr1⌬::KAN This study STT2 W303-1A THO2-TAP sub2⌬::HIS3 19 MTT1 W303-1A THO2-TAP mft1⌬::KAN S. Jimeno WTT4-5C W303-1A THO2-TAP hpr1-103 This study WHYL.2A W301-1A hpr1⌬::HIS3 GAL1pr::YRL454 19a 7452 HUERTAS ET AL. MOL.CELL.BIOL. on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from 8WG16 (Berkeley Antibody Company) and protein A-Sepharose for RNA polymerase II (RNAPII). The GFX purification system (Amersham) was used for the last DNA purification step. We used 20- to 27-bp oligonucleotides for PCR amplification of PMA1 sequences. For Tho2 recruitment, we amplified two fragments, localized at positions 292 to 321 and 2273 to 2242, whereas for Sub2 and RNAPII recruitment, we only amplified the 2273-to-2242 fragment. In both cases, we used the PCR of the intergenic region at positions 9716 to 9863 of chromosome V as a negative control. The PCR products obtained were electrophoresed in a 15% acrylamide gel, stained with ethidium bromide, and quantified in a Fuji FLA-3000. The relative abundance of each DNA fragment was calculated as the ratio of each DNA fragment to the intergenic-region quantification results of the precipitated fractions normalized with respect to the corresponding ratios of the input fractions. For analysis of RNAPII processivity, a strain harboring either YLR454 or lacZ under the control of the GAL1 promoter was used (23). RNAPII abundances at different positions of the genes were assayed as indicated, by using ChIP with anti-Rpb1-CTD monoclonal antibody 8WG16 and protein A- Sepharose as previously described (23). We used 20- to 30-bp oligonucleotides for PCR amplification of two fragments of YLR454, positions 3 to 43 and 7621 to 7674; of two fragments of lacZ, positions 715 to 1025 and 2247 to 2467; and of the 9716-to-9863 intergenic region of chromosome V that was used as a control. Real-time quantitative PCR was performed with SYBR green dye in the 7500 Real Time PCR system of Applied Biosystems by following the manufacturer’s instructions. Miscellanea. Recombination frequencies were calculated as previously described (18). For each genotype, the recombination frequencies are given as the average and standard deviation of the median recombination value obtained from two or three different transformants with 6 to 12 independent colonies per transformant. ␤-Galactosidase and phosphatase assays and Northern analyses were performed according to previously published procedures (18). mRNA export assays were performed by in situ poly(A) ⫹ RNA localization with a synthetic digoxigenin-labeled oligo(dT) 20 as previously described (5, 14). RESULTS Isolation of hpr1 mutations with differential effects on gene expression and genetic instability. Mutagenic PCR-generated hpr1 alleles were cloned into the YCp70 plasmid by in vivo homologous recombination (see Materials and Methods). hpr1 mutations that differentially affected gene expression and recombination were screened by a color assay based on the chromosomal leu2-k::ADE2-URA3::leu2-kdirect-repeat construct (4). With this construct, wild-type cells form white colonies (Ade ⫹ ) whereas hpr1 cells form red-sectoring colonies (Ade ⫹ Ade ⫺ ). Defects in gene expression were determined with a GAL1pr::lacZ fusion construct which results in a dark blue color in expression-competent wild-type colonies and a light blue color in expression-deficient hpr1 mutant colonies. Screening of 5,346 transformant colonies permitted the identification of four hpr1 mutants (hpr1-101 to hpr1-104) which, upon subsequent analyses, were verified as being preferentially hyperrecombinant or defective in lacZ expression. The hpr1 alleles were sequenced, and the mutations responsible for the amino acid changes of each allele were determined (Fig. 1A). hpr1-101 harbors two point mutations, but only one (L586P) leads to amino acid substitution. Interestingly, although the primary sequence of the Hpr1 protein is not particularly conserved, this mutation lies in a highly conserved region (Fig. 1B). hpr1-102 has a ⫺1 frameshift that leads to a premature stop codon and a carboxy-terminally truncated Hpr1 protein. hpr1-103 and hpr1-104 harbor multiple point mutations leading to 12 and 6 amino acid substitutions, respectively. Next, recombination frequencies and ␤-galactosidase activities were determined in a LAU3-10A hpr1⌬strain carrying the leu2-k::ADE2-URA3::leu2-kand GAL1pr::lacZ systems, respectively, and transformed with the YCp70::hpr1 series of plasmids (hpr1-101 to hpr1-104), YCpA13 (HPR1), or the empty vector YCp70 (hpr1) (Fig. 1C). hpr1⌬cells display a strong reduction in ␤-galactosidase activity (a 25-fold decrease) that correlates with a strong increase in recombination (773-fold above the wild-type level). Like hpr1⌬cells, hpr1-101 and hpr1-102 cells exhibit a significant reduction in ␤-galacto- sidase activity (4.3- and 6.7-fold decreases, respectively) which, in contrast to hpr1⌬, was accompanied by a weak increase in recombination (5- and 50-fold, respectively). Instead, ␤-galac- tosidase activity in hpr1-103 and hpr1-104 cells was poorly affected (1- to 2-fold decreases), while recombination values (239- and 567-fold above the wild-type level) were close to the values obtained in hpr1⌬. We therefore decided to further analyze hpr1-101 and hpr1-103 in order to understand the basis for the different phenotypes they produce. hpr1-101 causes a gene expression defect not associated with hyperrecombination, whereas hpr1-103 affects both gene expression and recombination. The strength of the gene expression defect and hyperrecombination phenotypes of hpr1⌬has been shown to be dependent on the DNA sequence (10). Thus, transcription through the lacZ bacterial gene is severely compromised in hpr1⌬and leads to strong hyperrecombination, but the transcription defect and hyperrecombination phenotypes are weak or barely detectable in other DNA sequences, such as that of PHO5. To understand the connection between the gene expression and recombination phenotypes observed in the hpr1 point mutants, we further analyzed the most representative alleles, i.e., hpr1-101 (strong gene expression defect and weak hyperrecombination) and hpr1-103 (weak gene expression defect and strong hyperrecombination). Thus, we constructed isogenic strains harboring single chromosomal copies of hpr1-101 (WH101-1A) and hpr1-103 (WH103-1A) at the HPR1 locus by gene replacement. Recombination analysis in the L-lacZ system, containing lacZ flanked by leu2 direct repeats, revealed that, compared to hpr1⌬(recombination 87-fold above the wild-type level), hyperrecombination was weak in hpr1-101 (8-fold) and moderate in hpr1-103 (20-fold) (Fig. 2A). Next, we analyzed recombination in the L-PHO5 and GL-lacZ systems, the latter of which is the same as L-lacZ except that transcription is driven by the GAL1 promoter. With either actively transcribed L-PHO5 or GL-lacZ under repressive conditions (2% glucose), we obtained wild-type recombination levels in the L-PHO5 and GL- lacZ systems in both hpr1-101 and hpr1-103, respectively. These results indicate that hyperrecombination was transcription and sequence dependent in both mutants. In order to determine whether transcription impairment and hyperrecombination in these mutants are associated, we analyzed transcript levels in the same system used to measure recombination. Northern analysis showed similar L-PHO5 transcript levels for all of the strains (Fig. 2B). Importantly, in the hpr1- 103 mutant the transcript levels of lacZ mRNA expressed from the L-lacZ system only reached 75 to 80% of the wild-type levels, which clearly differs from the ␤-galactosidase results obtained in the GAL1pr::lacZ system (Fig. 1C and data not shown). Thus, when transcription was analyzed in the same system in which hyperrecombination was detected, the hpr1- 103 mutant showed the same phenotypes as hpr1⌬, although to a lesser extent. As the L-lacZ and GAL1pr::lacZ systems differ in the length of the transcribed DNA sequence, it seems that VOL. 26, 2006 hpr1 SEPARATION-OF-FUNCTION MUTATION 7453 on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from the hpr1-103 effect on transcription was detectable in long DNA sequences. This is consistent with previous observations indicating that hyperrecombination is only observed in association with a detectable transcription defect (2). RNAPII processivity is impaired in hpr1-101 mutants. Since hpr1-101 did not display a significant hyperrecombination phenotype compared with hpr1⌬, we wondered whether this mutation leads to a real impairment in RNAPII transcription, as is the case for hpr1⌬strains. First we determined gene expression from GAL1pr::lacZ and GAL1pr::PHO5 in hpr1-101 mutant cells compared to wild-type and hpr1⌬cells. As shown in Fig. 3A, whereas ␤-galactosidase activity was clearly diminished in hpr1-101, acid phosphatase was not affected. This pattern is similar, although to a lesser extent, to the results obtained for the null hpr1⌬strain. Next, we determined whether the reduction of mRNA levels in hpr1-101,asinhpr1⌬, was different, depending on the gene tested. We analyzed the mRNA levels in various open reading frames (ORFs) driven by the strong GAL1 promoter. These ORFs included lacZ from Escherichia coli (3 kb and 53% GC content); PHO5 (1.5 kb and 40% GC), YAT1 (2 kb and 51% GC), and YRL454 (8 kb and 41% GC) from S.cerevisiae; and LAC4 (3 kb and 41% GC) from Kluyveromyces lactis. As shown in Fig. 3B, hpr1-101 causes a reduction as great as that produced by hpr1⌬in the mRNA levels of lacZ and YRL454 but has little effect on PHO5, consistent with the enzymatic analysis (Fig. 1). However, it has only a moderate effect on YAT1 and LAC4 mRNA levels. These results suggest that the strength of the deficiency in mRNA accumulation in hpr1-101 cells is also ORF dependent. In hpr1⌬strains, RNAPII processivity, defined as the ability of RNAPII to travel the entire length of a gene, is impaired (23). Although mRNA levels were affected in hpr1-101,itwas conceivable that RNAPII processivity was not impaired in this strain, therefore separating RNAPII processivity from hyperrecombination. To test this hypothesis, we determined the processivity of RNAPII in hpr1-101 along the lacZ and YRL454 genes by ChIP. This was performed with an anti-Rpb1 anti- FIG. 1. Genetic characterization of hpr1 point mutations that differentially affect transcription and recombination. (A) Schematic representation of the amino acid substitutions of the hpr1 alleles studied. Mutations that change the amino acid sequence are represented as black bars. (B) Protein alignment of the region surrounding L586 in several Hpr1 orthologs. The Hpr1 protein sequences used belong to the followings organisms: S.cerevisiae wild type and hpr1-101 mutant (Sc WT and Sc 101, respectively), Saccharomyces bayanus (SBay), Homo sapiens (Hsap), Mus musculus (Mmus), Xenopus laevis (Xlae), Brachydanio rerio (Brer), Drosophila melanogaster (Dmel), Saccharomyces kluyveri (Sklu), Caenorhabditis elegans (Cele), Arabidopsis thaliana (Atha), Dictyostelium discoideum (Ddis), Neurospora crassa (Ncra), and Schizosaccharomyces pombe (Spom). (C) ␤-Galactosidase activity and recombination frequencies of the LAU3-10A (hpr1⌬) strain containing the leu2-k::ADE2-URA3::leu2-k recombination system and transformed with pRS313-GZ (containing the GAL1pr:::lacZ fusion) and with either YCpA13 (HPR1), YCp-hpr1-101 to YCp-hpr1-104 (hpr1-101 to hpr1-104), or the empty vector YCp70. The percentage of ␤-galactosidase activity with respect to the wild-type level (taken as 100%) is shown. Each value represents the average of two or three independent experiments. The recombination frequencies (10 ⫺4 ) are averages of two to four median frequencies obtained from the same number of fluctuation experiments. 7454 HUERTAS ET AL. MOL.CELL.BIOL. on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from body and quantitative standard and real-time PCRs at the 5⬘ end and 3⬘end of each coding sequence. As shown in Fig. 4, RNAPII is significantly reduced toward the 3⬘end of lacZ in hpr1⌬, as well as hpr1-101 (Fig. 4A). In YRL454, RNAPII was significantly reduced at the 3⬘end in the hpr1⌬strain and to a lesser extent in the hpr1-101 strain (Fig. 4B). These results are consistent with the mRNA accumulation results observed for each gene and indicate that RNAPII processivity is diminished in hpr1-101. Thus, we conclude that impaired RNAPII processivity by itself is not sufficient to lead to hyperrecombination. Instead, in hpr1-103, RNAPII processivity along lacZ was not affected (Fig. 4B), in agreement with the expression data (Fig. 1C), but it was affected for the long (8-kb) YLR454 ORF to the same extent as in hpr1-101 (Fig. 4B). This is consistent with the conclusion that hpr1-103 affects transcription to a minor degree but sufficiently to cause transcription-dependent hyperrecombination. Nuclear accumulation of poly(A) ⴙ RNA in hpr1-101 and hpr1-103 mutants. The transcriptional defect of THO-null mutants such as hpr1⌬has been shown to be associated with nuclear accumulation of poly(A) ⫹ RNA caused by defects in mRNA export (19, 40). The functional relationship of THO with mRNA export is provided by the synthetic lethality of RNA export factor mutants in the absence of a functional THO complex, as demonstrated by the hpr1 mex67-5tsdouble mutants. To explore whether the hpr1-101 and -103 point mutants were affected in RNA export, we first determined the capacity of the new mutations to rescue the synthetic lethality of the hpr1⌬mex67-5mutant. For this, we constructed hpr1⌬ mex67-5strains carrying a URA3-based plasmid containing wild-type HPR1. These strains were transformed with a plasmid containing either mutant allele hpr1-101 or hpr1-103, and the capacity of each allele to rescue hpr1⌬mex67-5lethality was determined by growth in SC medium plus 5-fluoroacetic acid (FOA). As shown in Fig. 5A, the ability of each hpr1 allele to rescue hpr1 mex67-5lethality is linked to its transcriptional defect. hpr1-103, which had little effect on gene expression, almost completely rescued the synthetic lethality, whereas FIG. 2. Recombination in hpr1-101 and hpr1-103 mutants. (A) The isogenic strains W303-1A (WT), WH101-1A (hpr1-101), WH103-1A (hpr1-103), and SChY58a (hpr1⌬), carrying the mutant alleles integrated in the chromosomal HPR1 locus, were transformed with plasmid pSCh204 (L-lacZ recombination assay) or pSCh206 (L-PHO5 recombination assay). Gray boxes represent LEU2 repeats that flank either the PHO5 or the lacZ ORF, as indicated. The white box represents the promoter, and the white solid arrow represents the CYC transcription termination site. Shown are recombination frequencies under repressed conditions (glucose) in cells transformed with plasmid pRS314GLlacZ, containing the GL-lacZ recombination assay, in which transcription is under the control of the GAL1 promoter. The recombination frequencies shown are averages of two to four different experiments. (B) Northern analysis of the L-lacZ and L-PHO5 recombination system in isogenic strains W303-1A (WT [wild type]), WH101-1A (hpr1-101), and SChY58a (hpr1⌬) transformed with either pSCh204 or pSCh206, respectively. Filters were hybridized with either a lacZ oraPHO5 probe and with the 25S rRNA probe. All data were normalized with respect to the rRNA signal. The averages of two experiments are plotted. A.U., arbitrary units. VOL. 26, 2006 hpr1 SEPARATION-OF-FUNCTION MUTATION 7455 on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from hpr1-101, which confers strong transcription defects, did not, suggesting a direct correlation between transcription and impaired mRNA export. Analysis of other hpr1 alleles isolated in this study, such as hpr1-102 and hpr1-104, confirmed this correlation between transcription defect and synthetic lethality with mex67-5(data not shown). We next analyzed the nuclear export of poly(A) ⫹ mRNA by in situ hybridization with a 20-mer oligo(dT) fluorescent probe in the wild-type, hpr1-101,hpr1-103, and hpr1⌬strains (Fig. 5B). Incubation of the cells at 37°C led to nuclear poly(A) ⫹ mRNA accumulation in hpr1⌬and hpr1-101 cells. However, in hpr1-103 cells, mRNA accumulation was only observed after 6 h of incubation at 37°C. These results indicate that the gene expression defect in hpr1⌬and hpr1-101 cells directly correlates with their mRNA export defect. Transcription impairment in hpr1-101 and hyperrecombination in hpr1-103 are dependent on the nascent mRNA molecule. The transcription impairment and hyperrecombination phenotypes of hpr1⌬cells have been shown to be dependent on structures mediated by the nascent mRNA (18). To assay the FIG. 3. Transcription analysis of hpr1-101 versus the wild type and hpr1⌬as a function of GC content and the length of the transcribed DNA sequence. (A) Isogenic strains W303-1A (WT [wild type]), WH101-1A (hpr1-101), and SChY58a (hpr1⌬), carrying the mutant alleles integrated into the chromosomal HPR1 locus, were transformed with pRS316-GAL1lacZ (GAL1pr::lacZ fusion) or pSCh202 (GAL1p::PHO5 fusion). ␤-Galactosidase or phosphatase activity was defined as the percentage (for lacZ or PHO5, respectively) of the wild-type activity, which was taken as 100%. The mean and standard deviation of three independent experiments are plotted. (B) Northern analysis of mRNA levels in different plasmid constructs containing lacZ (pRS416GAL1lacZ), PHO5 (pSCH202), LAC4 (pSCH255), and YAT1 (pSCH247) under the control of the GAL1 promoter in strains W303-1A (wild type), WH101-1A (hpr1-101), and U768-4C (hpr1⌬). For the analysis of transcription of the YLR454 gene, strain WHYL.2A (hpr1⌬GAL1p::YLR454) was transformed with the empty vector YCpA70 (hpr1⌬) or with plasmid YCpA13 (HPR1)or YCp:hpr1-101 (hpr1-101). Others details are the same as those in Fig. 2B. 7456 HUERTAS ET AL. MOL.CELL.BIOL. on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from effect of the nascent mRNA on transcription, we used the previously defined Rib ⫹ and rib m constructs (Fig. 6A), in which a PHO5-Rib-lacZ transcriptional fusion containing either a wild-type (Rib ⫹ ) or a mutated (rib m ) Hammerhead ribozyme sequence was placed under the control of the GAL1 promoter (18). In both constructs, a 2.2-kb long mRNA is transcribed, but in the Rib ⫹ construct the active hammerhead ribozyme cleaves the transcript, liberating a 1.8-kb mRNA. Meanwhile, RNAPII travels farther, producing a 0.6-kb mRNA. Northern analysis of the rib m construct revealed that less transcript was accumulated in hpr1⌬and hpr1-101 cells than in wild-type and hpr1-103 cells. However, ribozyme cleavage in the Rib ⫹ construct led to an increase in the resulting 0.6-kb transcript so that similar mRNA levels were found in all strains. Apparently, ribozyme cleavage was capable of increasing the amount of mRNA transcripts in hpr1⌬and hpr1-101 cells. In a previous work, we showed that ribozyme cleavage was capable of partially suppressing the hyperrecombination phenotype observed in hpr1⌬cells. Thus, we asked whether ribozyme cleavage might also be sufficient to reduce the hyperrecombination phenotype observed in hpr1-103 cells. Hyperrecombination was assayed with the GL-Rib ⫹ and GL-rib m repeat systems containing PHO5, followed by active Rib ⫹ or inactive rib m sequences between 0.6-kb-long leu2 direct repeats, respectively (Fig. 6B). In the GL-rib m system, recombination was strongly increased in hpr1⌬(285-fold), moderately increased in hpr1- 103 (17-fold), and poorly affected in hpr1-101 (3-fold). In all three strains, recombination levels were reduced two- to threefold by the ribozyme-mediated cleavage of the nascent mRNA. Therefore, both the transcription defect of hpr1-101 and the hyperrecombination of hpr1-103 are similarly dependent on the nascent mRNA, as shown for hpr1⌬mutants. Differential stability and recruitment of the THO complex in hpr1-101 and hpr1-103 versus THO-null mutants. The THO complex is a highly stable complex which can be purified with salt concentrations as high as 1.2 M and remains stable in vivo in mutants of the TREX complex such as sub2⌬(9, 19). However, the integrity of the THO complex has not been studied in mutants of the THO subunits. To determine whether the different phenotypes of hpr1-101,hpr1-103, and the THO-null mutants were related to the stability of THO, the complex was purified from the different mutant strains. With a TAP epitope-tagged Tho2 protein, the complex was purified from whole cell extracts derived from wild-type, hpr1-101,hpr1-103, hpr1⌬,mft1⌬, and sub2⌬strains by two-step affinity purification (19, 33). Western blot analysis with a PAP antibody showed that the amount of TAP-Tho2 was similar in whole cell extracts of hpr1-101 and wild-type cells (Fig. 7A). Although TAP-Tho2 seems slightly reduced in hpr1-103 and sub2⌬and the amount of Tho2 varies between experiments (Fig. 7A), it was similar in all cases to that in wild-type cells. In contrast, in FIG. 4. RNA processivity is affected by hpr1-101 in a manner strongly dependent on the GC content and moderately dependent on the length of the transcribed DNA sequence. (A) ChIP assays of RNAPII at the lacZ gene. Experiments were performed with strains W303 (WT [wild type]), WH101-1A (hpr1-101), and U768-4C (hpr1⌬) transformed with plasmid pRS416GAL1lacZ containing lacZ under the control of the GAL1 promoter. ChIPs with regions 1 and 2 and an intergenic region as a control were performed in two different transformants, and PCRs were repeated four times for each transformant. The ratio of DNA in regions 1 and 2 was calculated from the DNA amounts in regions 1 and 2 relative to the DNA amount obtained from the intergenic region. Values were normalized to the amount of DNA in region 1 in each strain, which was set to 100% (for details, see Materials and Methods). (B) ChIP assays of RNAPII at the YLR454 gene under control of the GAL1 promoter. The experiments were performed with strain WHYL.2A (hpr1⌬GAL1p::YLR454) containing either plasmid YCpA13 (HPR1) or YCphpr1-101 (hpr1-101)orthe empty vector YCpA70 (hpr1⌬). ChIPs were performed with two different transformants each, and quantitative PCRs were repeated twice for each transformant. Values were normalized to the amount of DNA in region 1 of each transformant, which was set to 100%. VOL. 26, 2006 hpr1 SEPARATION-OF-FUNCTION MUTATION 7457 on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from hpr1⌬and mft1⌬, loading of similar amounts of cell extract onto the gel did not permit detection of a signal corresponding to the full TAP-Tho2 protein (Fig. 7A). Only when 10-fold more whole cell extract was loaded was a weak signal corresponding to the TAP-Tho2 protein detectable (Fig. 7A). As THO2 transcript levels have been found to be similar in wildtype and hpr1⌬cells (13), the decrease in Tho2 must result from a posttranscriptional effect, suggesting that THO subunits may only be stable in the form of a complex. Analysis of the TAP-Tho2 purified THO complex by gradient SDS-PAGE revealed that in the wild type, hpr1-101,hpr1- 103, and sub2⌬, the four THO subunits (Tho2, Hpr1, Mft1, and Thp2) were present (Fig. 7B). However, in hpr1⌬and mft1⌬, only the TAP-Tho2 protein was enriched by the TAP system. Thus, if one of the core proteins of THO is missing, the integrity of the THO complex is greatly reduced. In order to find out whether the remaining proteins form at least some kind of complex, we used an anti-Mft1 antibody to detect FIG. 5. mRNA export defects of hpr1 mutants. (A) Suppression of hpr1⌬mex67-5synthetic lethality. Strain WMH1 (hpr1⌬mex67-5) transformed with URA3-based plasmid pRS316-HPR1 was additionally transformed with LEU2-based plasmids YCpA13 (HPR1), YCp-hpr1-101 (hpr1-101), and YCp-hpr1-103 (hpr1-103) or the empty vector YCp70 (hpr1⌬). Transformants were spotted in 10-fold serial dilutions on selective medium with or without FOA. Plates were scanned after 3 days of incubation at 30°C. Note that only transformants harboring alleles suppressing the synthetic lethality are able to grow on SC medium-FOA plates. (B) Analysis of nuclear poly(A) ⫹ RNA localization in W303-1A (wild type), WH101-1A (hpr1-101), WH103-1A (hpr1-103), and U768-4C (hpr1⌬) log-phase cultures cultivated for 3 or6hat37°C, as indicated. Samples were immobilized in Teflon-coated slides and hybridized first with a digoxigenin-labeled 20-mer oligo(dT) and second with a fluorescein-conjugated anti-digoxigenin antibody for in situ detection of mRNA. Nuclei were stained with 4⬘,6⬘-diamidino-2-phenylindole (DAPI). U, uracil; L, leucine. 7458 HUERTAS ET AL. MOL.CELL.BIOL. on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from remaining traces of Mft1 in the TAP-Tho2 purified samples. Similar amounts of Mft1 were detected in wild-type, hpr1-101, hpr1-103, and sub2⌬cells, whereas no signal was detected in hpr1⌬and mft1⌬cells when similar amounts of TAP-purified Tho2 were loaded (Fig. 7C). However, when 10-fold more Tho2 protein was loaded onto the gel, Mft1 could be detected in hpr1⌬TAP-Tho2 purified fractions (Fig. 7C). These results indicate that the THO complex is unstable if one of its components is absent, such as Hpr1 or Mft1, but is stable in both the hpr1-101 and hpr1-103 mutants, as well as in mutants in which a TREX component, such as Sub2, is absent. Since, contrary to hpr1⌬, THO is stable in hpr1-101 and hpr1-103 cells, we wondered if THO was also recruited into active chromatin in these mutants. With the same TAP-Tho2 strains as before, we found that hpr1-101 and hpr1-103 recruited the TAP-Tho2 subunit of THO to a transcriptionally active PMA1 gene (Fig. 8) at levels clearly above that of hpr1⌬ mutants. Therefore, the two point mutations analyzed are not null alleles and form THO complexes that are competent for chromatin recruitment. Sub2 is poorly recruited to active chromatin in both hpr1- 101 and hpr1-103 mutants. The facts that the presence of THO at the site of transcription elongation helps recruit Sub2 to transcribed chromatin (1, 46) and that multicopy SUB2 is able to suppress hpr1⌬(11, 19) are consistent with the hypothesis that Sub2 recruitment to the nascent mRNA plays a key role in gene expression. As THO is as stable in both hpr1-101 and hpr1-103 mutants as in wild-type cells, we wondered whether their respective phenotypes could be caused by different levels of Sub2 recruitment to transcribed chromatin. While testing this possibility, we first noticed that with a lacZ-URA3 translational fusion under the control of the tet promoter (LAUR system) (19), SUB2 overexpression suppressed the transcriptional defects of both the hpr1-101 and hpr1-103 mutants (Fig. 9A). This was consistent with the possibility that Sub2 was not efficiently recruited to transcribed chromatin in both mutants. Again, hpr1-103 showed a transcription defect in extra-long DNA sequences (Fig. 9A) that reinforces the conclusion that hpr1-103 is a leaky rather than a separation-of-function mutation. Sub2 recruitment to active chromatin was directly assayed by ChIP in strains carrying a SUB2-TAP epitope fusion at the SUB2 chromosomal locus with Sepharose IgG and anti- RNAPII antibodies. As shown in Fig. 9B, there was no significant difference in the recruitment of RNAPII to the constitutively transcribed PMA1 gene between the wild-type and hpr1 mutant strains tested. Nevertheless, recruitment of Sub2-TAP was as inefficient in hpr1-101 and hpr1-103 cells as in hpr1⌬ cells (55 to 60% of the wild-type level). Therefore, we can conclude that the specific gene expression phenotype of hpr1- FIG. 6. Nascent-mRNA dependency of the transcription and hyperrecombination defects of hpr1 mutants. (A) Kinetic analyses of transcription activation of the rib m and Rib ⫹ fusions in strains W303-1A (WT [wild type]), WH101-1A (hpr1-101), WH103-1A (hpr1- 103), and U678-4C (hpr1⌬). The PHO5-rib m -lacZ (rib m ) and PHO5- Rib ⫹ -lacZ (Rib ⫹ ) transcriptional fusions were under control of the GAL1 promoter (GAL1 p ). They contain an active or inactive (respectively), synthetically made, 52-bp ribozyme (Rib), followed by a 266-bp fragment of the U3 gene to prevent the cleaved mRNA from degradation and the 369-bp PvuII 3⬘-end lacZ fragment at the untranslated region of PHO5 (position ⫹1405). Samples were collected 2 h after galactose addition to activate transcription. Electrophoresis was performed with formaldehyde-agarose gels (rib m ) or urea-acrylamide gels (Rib ⫹ ), and hybridization was with a U3 probe, corresponding to the rib m and Rib ⫹ region in the scheme. All data were normalized with respect to the endogenous U3 signal. The average of three different experiments is plotted. A.U., arbitrary units. (B) Recombination frequencies of wild-type, hpr1,hpr1-101, and hpr1-103 cells containing the recombination systems GL-Rib ⫹ (black bars) and GL-rib m (gray bars) are shown. Each recombination frequency is the median value of six independent colonies. The average median value of two to four experiments and the standard deviation are plotted. VOL. 26, 2006 hpr1 SEPARATION-OF-FUNCTION MUTATION 7459 on June 29, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://mcb.asm.org/Downloaded from