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• W1973607544 abstract "THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1Δ, tho2Δ, and mft1Δ mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1Δ mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export. THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1Δ, tho2Δ, and mft1Δ mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1Δ mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export. mRNA synthesis in eukaryotes is a multistep process mediated by RNA polymerase II (RNAPII) 1The abbreviations used are: RNAPII, RNA polymerase II; mRNP, mRNA and heterogeneous nuclear ribonucleoprotein complex; WCE, whole cell extract; WT, wild-type; nt, nucleotide.1The abbreviations used are: RNAPII, RNA polymerase II; mRNP, mRNA and heterogeneous nuclear ribonucleoprotein complex; WCE, whole cell extract; WT, wild-type; nt, nucleotide. and consists of three major stages, i.e. initiation, elongation, and termination. During elongation, RNAPII has to overcome situations derived from transient pausing caused by regulatory signals with the help of transcriptional elongation factors. These factors associate with RNAPII to facilitate elongation through either particular DNA sequences or chromatin (1Shilatifard A. Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 2003; 72: 693-715Crossref PubMed Scopus (198) Google Scholar, 2Hartzog G.A. Curr. Opin. Genet. Dev. 2003; 13: 119-126Crossref PubMed Scopus (40) Google Scholar). Among these factors, there is functional evidence for roles in transcription elongation for TFIIS (3Sekimizu K. Kobayashi N. Mizuno D. Natori S. Biochemistry. 1976; 15: 5064-5070Crossref PubMed Scopus (65) Google Scholar, 4Wind M. Reines D. BioEssays. 2000; 22: 327-336Crossref PubMed Scopus (168) Google Scholar), TFIIF (5Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (116) Google Scholar, 6Lei L. Ren D. Burton Z.F. Mol. Cell. Biol. 1999; 19: 8372-8382Crossref PubMed Scopus (24) Google Scholar), human elongin (7Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (292) Google Scholar), human 11-19 lysine-rich leukemia (ELL) (8Shilatifard A. Lane W.S. Jackson K.W. Conaway R.C. Conaway J.W. Science. 1996; 271: 1873-1876Crossref PubMed Scopus (279) Google Scholar), human FACT/yeast Spt16-Pob3 (9Orphanides G. LeRoy G. Chang C.H. Luse D.S. Reinberg D. Cell. 1998; 92: 105-116Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 10LeRoy G. Orphanides G. Lane W.S. Reinberg D. Science. 1998; 282: 1900-1904Crossref PubMed Scopus (256) Google Scholar, 11Wada T. Orphanides G. Hasegawa J. Kim D.K. Shima D. Yamaguchi Y. Fukuda A. Hisatake K. Oh S. Reinberg D. Handa H. Mol. Cell. 2000; 5: 1067-1072Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), human DSIF/yeast Spt4-Spt5 (12Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar, 13Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (371) Google Scholar), human NELF (14Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar), and the 19 S proteasome subunit (15Ferdous A. González F. Sun L. Kodadek T. Johnston S.A. Mol. Cell. 2001; 7: 981-991Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). In addition, there is a set of eukaryotic factors that might also have an effect in transcription elongation. One such factor is THO/TREX. THO was identified in yeast as a four-protein complex containing Tho2, Hpr1, Mft1, and Thp2 (16Chávez S. Beilharz T. Rondón A.G. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Lithgow T. Aguilera A. EMBO J. 2000; 19: 5824-5834Crossref PubMed Scopus (243) Google Scholar). Null mutations in each of the genes encoding the subunits of THO confer increased recombination between direct repeats, high levels of plasmid and chromosome instability, and defects in gene expression. This is particularly evident for long and GC-rich DNA sequences such as lacZ (16Chávez S. Beilharz T. Rondón A.G. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Lithgow T. Aguilera A. EMBO J. 2000; 19: 5824-5834Crossref PubMed Scopus (243) Google Scholar, 17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar, 18Chávez S. García-Rubio M. Prado F. Aguilera A. Mol. Cell. Biol. 2001; 21: 7054-7064Crossref PubMed Scopus (98) Google Scholar, 19Piruat J.I. Aguilera A. EMBO J. 1998; 17: 4859-4872Crossref PubMed Scopus (123) Google Scholar). The observation that transcription of some DNA sequences is impaired in THO mutants and that hyper-recombination only occurs at actively transcribed sequences whose transcription is THO-dependent suggests that transcription elongation is impaired in these mutants (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar, 19Piruat J.I. Aguilera A. EMBO J. 1998; 17: 4859-4872Crossref PubMed Scopus (123) Google Scholar, 20Prado F. Piruat J.I. Aguilera A. EMBO J. 1997; 16: 2826-2835Crossref PubMed Scopus (80) Google Scholar). However, in contrast to mutants of bona fide transcription elongation factors, THO mutants show transcription-dependent genetic instability and are not sensitive to 6-azauracil (6-AU) (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar), a hallmark phenotype associated with transcription elongation defects (21Shaw R.J. Reines D. Mol. Cell. Biol. 2000; 20: 7427-7437Crossref PubMed Scopus (113) Google Scholar). Recently, THO has been shown to be present in a larger complex, termed TREX, together with components of the mRNA export machinery such as Sub2 and Yra1 (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar). In addition, sub2 and yra1 mutants show the same gene expression defects and transcription-associated hyper-recombination as those of the THO mutants (23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar, 24Fan H.Y. Merker R.J. Klein H.L. Mol. Cell. Biol. 2001; 21: 5459-5470Crossref PubMed Scopus (49) Google Scholar). This, together with the mRNA export defect of THO mutants, the THO-like gene expression and recombination phenotypes of mutants of the Mex67-Mtr2 mRNA export factor, and the ability of THO to bind RNA in vitro, suggests that THO might be functionally involved in mRNP biogenesis and export (23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar). This conclusion is strengthened by the observation that mutants of the Thp1·Sac3 complex, which interacts with RNA and the nuclear pore complex (NPC) and participates in mRNA export (25Gallardo M. Aguilera A. Genetics. 2001; 157: 79-89PubMed Google Scholar, 26Fischer T. Strasser K. Racz A. Rodríguez-Navarro S. Oppizzi M. Ihrig P. Lechner J. Hurt E. EMBO J. 2002; 21: 5843-5852Crossref PubMed Scopus (222) Google Scholar), confer the same transcription and hyper-recombination phenotypes as do THO/TREX mutations (25Gallardo M. Aguilera A. Genetics. 2001; 157: 79-89PubMed Google Scholar, 27Gallardo M. Luna R. Erdjument-Bromage H. Tempst P. Aguilera A. J. Biol. Chem. 2003; 278: 24225-24232Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Sub2 is a conserved eukaryotic protein of the family of the DEAD box RNA helicases. First identified as a protein functioning at multiple steps during spliceosome assembly (28Libri D. Graziani N. Saguez C. Boulay J. Genes Dev. 2001; 15: 36-41Crossref PubMed Scopus (111) Google Scholar, 29Zhang M. Green M.R. Genes Dev. 2001; 15: 30-35Crossref PubMed Scopus (91) Google Scholar, 30Kistler A.L. Guthrie C. Genes Dev. 2001; 15: 42-49Crossref PubMed Scopus (165) Google Scholar), Sub2 and its human (hUAP56) and Drosophila (HEL) orthologues have also been involved in mRNA transport (31Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (304) Google Scholar, 32Strasser K. Hurt E. Nature. 2001; 413: 648-652Crossref PubMed Scopus (222) Google Scholar, 33Jensen T.H. Boulay J. Rosbash M. Libri D. Curr. Biol. 2001; 11: 1711-1715Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 34Gatfield D. Le Hir H. Schmitt C. Braun I.C. Kocher T. Wilm M. Izaurralde E. Curr. Biol. 2001; 11: 1716-1721Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Indeed, Sub2 binds in vivo to Yra1, an RNA binding protein (35Strasser K. Bassler J. Hurt E. J. Cell Biol. 2000; 150: 695-706Crossref PubMed Scopus (183) Google Scholar) with RNA-RNA annealing activity (36Portman D.S. O'Connor J.P. Dreyfuss G. RNA. 1997; 3: 527-537PubMed Google Scholar). Yra1 interacts with the Mex67-Mtr2 mRNA export factor (32Strasser K. Hurt E. Nature. 2001; 413: 648-652Crossref PubMed Scopus (222) Google Scholar, 37Zenklusen D. Vinciguerra P. Wyss J.C. Stutz F. Mol. Cell. Biol. 2002; 22: 8241-8253Crossref PubMed Scopus (254) Google Scholar), a heterodimer that mediates the interaction of the mRNP with the nuclear pore complex (35Strasser K. Bassler J. Hurt E. J. Cell Biol. 2000; 150: 695-706Crossref PubMed Scopus (183) Google Scholar, 38Santos-Rosa H. Moreno H. Simos G. Segref A. Fahrenkrog B. Pante N. Hurt E. Mol. Cell. Biol. 1998; 18: 6826-6838Crossref PubMed Scopus (221) Google Scholar). A number of observation suggest that THO may form a core complex apart from Sub2. First, removal of Sub2 in cells does not affect the integrity of the purified THO complex (23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar). Second, in a highly purified THO complex, Sub2 is a minor component and Yra1 is absent (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar). Finally, there is an important part of Sub2 in the cell that is not in association with the THO complex, as observed in purification experiments using tandem affinity purification (TAP)-tagged Sub2 (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar). Despite the cumulative in vivo evidence indicating that THO and Sub2 mutants confer pleiotropic phenotypes on transcription and mRNA metabolism (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar, 18Chávez S. García-Rubio M. Prado F. Aguilera A. Mol. Cell. Biol. 2001; 21: 7054-7064Crossref PubMed Scopus (98) Google Scholar, 22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar, 23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar), no direct in vitro proof exists yet that transcription elongation is defective in these mutants. To determine whether mRNA elongation was defective in mutants of the THO/TREX complex, we performed in vitro transcription elongation assays with two G-less cassette DNA templates (39Rondón A.G. García-Rubio M. González-Barrera S. Aguilera A. EMBO J. 2003; 22: 612-620Crossref PubMed Scopus (79) Google Scholar). In this study, we show that cell extracts of THO complex mutants (hpr1Δ, tho2Δ, and mft1Δ) and sub2 have significantly reduced transcription elongation efficiencies in vitro. In addition, we demonstrate that the decrease in mRNA accumulation of hpr1Δ mutants is not due to an increase in mRNA decay. It is likely that THO is not a bona fide transcription elongation factor but rather a protein complex involved in mRNP biogenesis during transcription elongation. Yet, our work provides unequivocal molecular evidence that transcription elongation efficiency is reduced in THO/TREX mutants, which is in agreement with the idea that transcription and mRNA metabolism and export are coupled in the cell. Yeast Strains and Plasmids—Strains used were wild-type (W303-1A) and the isogenic mutants tho2Δ::KAN (RK2-6D) (19Piruat J.I. Aguilera A. EMBO J. 1998; 17: 4859-4872Crossref PubMed Scopus (123) Google Scholar), hpr1Δ::KAN (SChY58-a), hpr1Δ::HIS3 (U678-1C) (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar), mft1Δ::KAN (WMK-2A) (16Chávez S. Beilharz T. Rondón A.G. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Lithgow T. Aguilera A. EMBO J. 2000; 19: 5824-5834Crossref PubMed Scopus (243) Google Scholar), sub2Δ::HIS3 (Ura- segregant of DLY23), and sub2-201 (DLY33/sub2-201) (28Libri D. Graziani N. Saguez C. Boulay J. Genes Dev. 2001; 15: 36-41Crossref PubMed Scopus (111) Google Scholar). We have also used wild-type BY4741 and its isogenic deletion rrp6Δ::KAN (YOR001w) (Euroscarf, Frankfurt, Germany). Strains HRK-1A (WT), HRK-2B (rrp6Δ::KAN), HRK-3C (hpr1Δ::HIS3), and HRK-44C (hpr1Δ::HIS3 rrp6Δ::KAN) come from genetic crosses between U678-1C and YOR001w. His6-tagged Gal4-VP16 recombinant protein was expressed in Escherichia coli from plasmid pRJRGAL4-VP16 (M. Ptashne, Sloan-Kettering Institute, New York, NY). We used plasmid pGCYC1-402 (39Rondón A.G. García-Rubio M. González-Barrera S. Aguilera A. EMBO J. 2003; 22: 612-620Crossref PubMed Scopus (79) Google Scholar) for in vitro transcription elongation assays and p416GAL1lacZ (40Mumberg D. Muller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (796) Google Scholar) for expression analysis. Preparation of Yeast Whole Cell Extracts (WCEs)—Yeast cells were grown in rich YEPD (1% yeast extract, 2% peptone, and 2% dextrose) medium at 30 °C to an A 600 of 1. WCEs were prepared as described (41Schultz M.C. Choe S.Y. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1004-1008Crossref PubMed Scopus (83) Google Scholar), with the exception that the extraction buffer was 0.2 m Tris, pH 7.5, 0.39 m ammonium sulfate, 10 mm MgSO4, 20% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2.7 mm benzamidine, 0.3 μg/ml leupeptin, and 1.4 μg/ml pepstatin A. Precipitation and dialysis were performed as described (42Woontner M. Wade P.A. Bonner J. Jaehning J.A. Mol. Cell. Biol. 1991; 11: 4555-4560Crossref PubMed Scopus (79) Google Scholar). WCEs were distributed in aliquots and frozen in liquid nitrogen. They were stable after repeated cycles of freezing and thawing. In Vitro Transcription Assay with Whole Cell Extracts—Each reaction was performed in a final volume of 40 μl of buffer A 0.5 (20 mm HEPES, pH 7.5, 20% glycerol, 1 mm EDTA, 1 mm dithiothreitol, and 500 mm potassium acetate) with 100 μg of whole cell extracts and 100 ng of Gal4-VP16 purified as described (43Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar) and dialyzed in buffer A 0.05 (same as A 0.5 but with 50 mm potassium acetate). Final potassium acetate concentration should be <150 mm. The reaction was set up adding 20 μl of transcription buffer (2×) (final concentration was 40 mm HEPES-KOH, pH 7.5, 5 mm MgCl2, 1 mm ATP, 1 mm GTP, 0.5 mm UTP, 0.03 mm CTP, 40 mm phosphocreatine, 32 μg of creatine kinase, 5 mm dithiothreitol, and 7.5 units of the RNase inhibitor RNAguard (Amersham Biosciences)). After 20 min of preincubation at room temperature, 400 ng of pGCYC402 and 1 μl of [α32P]CTP (3000 Ci/mmol) were added. The reaction was stopped at indicated times by the addition of 200 μl of stop buffer (10 mm Tris-HCl, pH 7.5, 0.3 m NaCl, and 5 mm EDTA) and 200 units of RNaseT1 for 15 min at room temperature. Samples were treated with proteinase K, which was phenol-extracted and run in a sequencing gel as described (44Sayre M.H. Tschochner H. Kornberg R.D. J. Biol. Chem. 1992; 267: 23383-23387Abstract Full Text PDF PubMed Google Scholar). The amount of radioactivity incorporated was quantified with a Fuji FLA3000. In Vivo Analysis of Gene Expression and mRNA Decay—Ten micrograms of total RNA was prepared from induced cultures and used for Northern analysis following standard procedures (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar). DNA filters were hybridized first with 32P-labeled DNA probes as specified. For determination of the total amount of RNA used, filters were stripped and re-hybridized with 32P-labeled 25 S rDNA obtained by PCR as described (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar). Miscellaneous—DNA isolation, 32P-radiolabeling, genetic crosses, and yeast transformations were performed according to standard procedures. Whole Cell Extracts of Mutants of the THO Complex Are Impaired in Transcription Elongation—To determine whether mutants of the THO complex were impaired in transcription elongation, we used our newly reported in vitro assay (39Rondón A.G. García-Rubio M. González-Barrera S. Aguilera A. EMBO J. 2003; 22: 612-620Crossref PubMed Scopus (79) Google Scholar). This assay is based on a plasmid (pGCYC1-402) in which a hybrid GAL4-CYC1 promoter containing a Gal4 binding site is fused to a 1.88-kb DNA fragment coding two G-less cassettes separated by 1.4 kb. The first G-less cassette is right downstream of the promoter and is 84-nt-long. The second is located at 1.48 kb from the promoter and is 376-nt-long. In this assay, transcription activated by Gal4-VP16 leads to an mRNA that, after digestion with RNase T1, which degrades all G-containing mRNA sequences, leaves the two G-less cassettes intact (Fig. 1A). The efficiency of transcription elongation is determined in WCEs by the values of the ratio of accumulation of the 376-versus the 84-nt-long G-less RNA fragments. We have recently proved with this assay that transcription elongation is defective in spt4Δ WCE (39Rondón A.G. García-Rubio M. González-Barrera S. Aguilera A. EMBO J. 2003; 22: 612-620Crossref PubMed Scopus (79) Google Scholar). As can be seen in Fig. 1B, after 30 min of transcription tho2Δ, hpr1Δ, and mft1Δ cell extracts carrying deletions of structural genes of the THO complex fully transcribed the 376-nt G-less cassette with efficiencies ranging from 58 to 71% of the wild-type levels. These results indicate that removal of any subunit of the THO complex leads to a similar reduction in the efficiency of transcription elongation in vitro. To determine whether or not the addition of Gal4-VP16 could affect transcription elongation in wild-type and THO mutants differently, we repeated the experiments in the absence and the presence of externally added Gal4-VP16. We first showed that, as expected, transcription initiation efficiency in the absence of Gal4-VP16, as determined by the accumulation of the 84-nt G-less cassette, was lower (22%) than in the presence of Gal4-VP16. Furthermore, as can be seen in Fig. 1C, the addition of Gal4-VP16 diminished the efficiency of transcription elongation similarly in hpr1Δ and wild-type cells. Therefore, the lower efficiency of transcription elongation observed in the THO mutants is independent of the transcription activator. To further demonstrate that elongation was less efficient in mutants of the THO complex, we determined the kinetics of transcription elongation in time course experiments in mutants of the two largest subunits of the complex, tho2Δ and hpr1Δ. As can be seen in Fig. 2, transcription elongation efficiency in both mutant extracts was 46-66% of the wild-type, depending on the time the reaction was run. We conclude that THO complex depletion causes a deficiency of transcription elongation in whole cell extracts. It is worth noting that this deficiency is independent of the growth rate of the strains tested, because whereas mft1Δ cells grow like wild-type cells and hpr1Δ cells grow poorly, both mutations have similar effects on transcription elongation. Whole Cell Extracts of Mutants of Sub2 Show Transcription Elongation Impairment—The THO complex is associated with proteins involved in mRNA export, such as Sub2 and Yra1, in a larger complex termed TREX. However, THO may form a core complex apart from Sub2 (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar). This, together with the fact that Sub2 and Yra1 are proteins with functional roles in mRNA splicing and/or mRNA export, makes it important to know whether Sub2 is also required for efficient transcription elongation. We applied in vitro transcription analysis to sub2 mutants. As can be seen in Fig. 1B, after 30 min of transcription, sub2Δ WCEs fully transcribed the 376-nt G-less cassette with 36% of the efficiency of the wild-type. To get further evidence of the role of Sub2 in transcription elongation, we determined the kinetics of transcription elongation of whole cell extracts with time course experiments using WCEs of the sub2-201 mutant. It has been reported that the sub2-201 mutation causes a splicing phenotype at 37 °C in WCEs in vitro that is not observed at the permissive temperature (28Libri D. Graziani N. Saguez C. Boulay J. Genes Dev. 2001; 15: 36-41Crossref PubMed Scopus (111) Google Scholar). Elongation assays were performed by maintaining the WCEs at 23 °C (unheated) or shifting them to 37 °C for 15 min (heated) immediately before starting the experiment. As can be seen in Fig. 3, transcription elongation was reduced in unheated WCEs (54% of the wild-type levels after 40 min), indicating that sub2-101 was leaky at the permissive temperature for transcription elongation. However, the reduction in transcription elongation efficiency was stronger in heated sub2-201 WCEs in which the elongation levels were similar to those of sub2Δ cells (30% of the wild-type levels). This result indicates that Sub2 is required for efficient elongation. The Gene Expression Defect of hpr1Δ Is Not Due to an Increase in mRNA Decay—Recently, the question has been raised of whether the low mRNA levels observed in vivo in mutants of the THO/TREX complex could be due to a higher rate of mRNA degradation and not to a transcription defect (37Zenklusen D. Vinciguerra P. Wyss J.C. Stutz F. Mol. Cell. Biol. 2002; 22: 8241-8253Crossref PubMed Scopus (254) Google Scholar, 45Libri D. Dower K. Boulay J. Thomsen R. Rosbash M. Jensen T.H. Mol. Cell. Biol. 2002; 22: 8254-8266Crossref PubMed Scopus (214) Google Scholar). It was previously observed that steady-state levels of lacZ mRNA driven from a GAL10 promoter in yra1-8 rrp6Δ mutants were ∼2-3-fold higher than those in yra1-8 mutants (37Zenklusen D. Vinciguerra P. Wyss J.C. Stutz F. Mol. Cell. Biol. 2002; 22: 8241-8253Crossref PubMed Scopus (254) Google Scholar). Also, the 5′ end of HSP104 mRNA accumulates ∼2 times above the 3′-end in hpr1Δ and sub2-201 single mutants but not in hpr1Δ rrp6Δ and sub2-201 rrp6Δ double mutants (45Libri D. Dower K. Boulay J. Thomsen R. Rosbash M. Jensen T.H. Mol. Cell. Biol. 2002; 22: 8254-8266Crossref PubMed Scopus (214) Google Scholar). However, these differences are very low as compared with the 20-fold decrease in expression of genes such as lacZ observed in time course experiments (16Chávez S. Beilharz T. Rondón A.G. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Lithgow T. Aguilera A. EMBO J. 2000; 19: 5824-5834Crossref PubMed Scopus (243) Google Scholar) and do not explain the gene expression and recombination phenotypes of THO mutants. We decided to assay whether the gene expression defects of THO/TREX mutants reflected transcription defects rather than higher mRNA degradation rates. We measured the half-life of two differently affected mRNAs in THO mutants, i.e. the lacZ and GAL1 mRNAs, both driven from the GAL1 promoter. It was shown previously that lacZ was poorly expressed in vivo in THO mutants, whereas GAL1 levels of expression were proximal to wild-type levels (16Chávez S. Beilharz T. Rondón A.G. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Lithgow T. Aguilera A. EMBO J. 2000; 19: 5824-5834Crossref PubMed Scopus (243) Google Scholar, 17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar). As can be seen in Fig. 4, A and B, hpr1Δ and wild-type cells have similar kinetics of degradation of both lacZ and GAL1 mRNAs. Indeed, we do not detect higher levels of mRNA stability in rrp6Δ mutants (Fig. 4C). That is, mRNA stability is the same in both wild-type and mutant strains, regardless of whether or not the expression of the mRNA analyzed was affected by hpr1Δ. Therefore, failure of the hpr1Δ mutant and, by extension, THO/TREX mutants to accumulate mRNAs such as lacZ in vivo (17Chávez S. Aguilera A. Genes Dev. 1997; 11: 3459-3470Crossref PubMed Scopus (145) Google Scholar) was not due to an increase in mRNA decay but to low levels of transcript formation. This is consistent with the transcription elongation defect detected in vitro. The prediction of this result is that rrp6Δ should not significantly suppress the defect in the kinetics of accumulation of lacZ mRNA caused by hpr1Δ. As expected, Fig. 5 shows similar kinetics of transcription in hpr1Δ and hpr1Δ rrp6Δ strains that, in both strains, occur at 42 and 22% of the wild-type levels after 1 h of induction, respectively.Fig. 5Expression analysis of GAL1pr::lacZ construct in HRK-1A (WT), HRK-2B (hpr1Δ), and HRK-44C (hpr1Δ rrp6Δ) congenic strains. A, β-galactosidase activity of strains transformed with plasmid p416GAL1lacZ. Each value represents the average of two different transformants. Only the data of induced expression (2% galactose) are given. Under repression conditions (2% glucose), values were below detection levels in all cases. U, units. B, Northern analysis of lacZ mRNA driven from the GAL1 promoter and GAL1 endogenous locus. Mid-log phase cultures of cells transformed with plasmid p416GAL1lacZ were diluted in synthetic complete (SC) 3% glycerol-2% lactate media lacking uracil to an A 600 of 0.5 and incubated for 16 h at 30 °C. 2% galactose was then added, and samples were taken at different times, as specified. Other details are as described in the Fig. 4 legend. AU, arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using an in vitro transcription elongation assay based on supercoiled DNA templates containing two G-less cassettes, we provide molecular evidence that the THO/TREX complex, with a role at the interface between transcription and mRNA export, is required for transcription elongation. The different mutants of the THO core complex (hpr1Δ, tho2Δ, and mft1Δ) and sub2Δ and sub2-201 showed inefficient transcription elongation in vitro, consistent with previously reported in vivo mRNA accumulation defects. We excluded experimentally the possibility that the mRNA expression defect of hpr1Δ mutants and, by extension, THO/TREX mutants, was due to high mRNA degradation rates. Several results suggest that THO has a role in mRNP biogenesis and export. First, the tandem affinity purification-tagged THO complex copurified with mRNA export proteins Sub2 and Yra1 (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar). Second, mutants of the mRNA export factors Sub2, Yra1, Mex67, Mtr2, Thp1, and Sac3 showed transcription-dependent hyper-recombination and gene expression phenotypes like those of THO mutants (23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar, 24Fan H.Y. Merker R.J. Klein H.L. Mol. Cell. Biol. 2001; 21: 5459-5470Crossref PubMed Scopus (49) Google Scholar, 25Gallardo M. Aguilera A. Genetics. 2001; 157: 79-89PubMed Google Scholar,27Gallardo M. Luna R. Erdjument-Bromage H. Tempst P. Aguilera A. J. Biol. Chem. 2003; 278: 24225-24232Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Third, THO mutants are affected in mRNA export (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar, 46Schneiter R. Guerra C.E. Lampl M. Gogg G. Kohlwein S.D. Klein H.L. Mol. Cell. Biol. 1999; 19: 3415-3422Crossref PubMed Google Scholar). It was therefore important to prove that the gene expression defect observed in mutants of the THO complex was the result of an impairment of transcription elongation. Our in vitro transcription elongation assay unequivocally shows that, regardless of its particular function, THO is required for efficient transcription elongation. In an assay designed to specifically quantify transcription elongation efficiency, the hpr1Δ, tho2Δ, and mft1Δ mutants of the THO complex reduced the efficiency of transcription elongation to 58-71% of the wild-type levels (Figs. 1 and 2). THO does not have an effect on the rate of mRNA decay that could explain the differences of gene expression of THO mutants (Figs. 4 and 5). Degradation rates of mRNAs are the same in hpr1Δ and wild-type mutants, regardless of whether or not expression of the gene studied (lacZ or GAL1) was negatively affected by hpr1Δ (Fig. 4). In addition, rrp6Δ, a null mutation of the Rrp6 exoribonuclease component of the nuclear exosome, does not significantly suppress the lacZ expression defect caused by hpr1Δ. As expected from this result, rrp6Δ does not suppress the hyper-recombination phenotype of hpr1Δ mutants. 2P. Huertas and A. Aguilera, unpublished results. Our mRNA stability results are consistent, therefore, with the in vitro analysis showing a defect in transcription elongation and with the hallmark phenotype of THO mutants, a transcription elongation-dependent hyper-recombination. Sub2 is a putative RNA helicase of the DEAD box family that has been shown to have a role in mRNA splicing and export (28Libri D. Graziani N. Saguez C. Boulay J. Genes Dev. 2001; 15: 36-41Crossref PubMed Scopus (111) Google Scholar, 29Zhang M. Green M.R. Genes Dev. 2001; 15: 30-35Crossref PubMed Scopus (91) Google Scholar, 31Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (304) Google Scholar, 32Strasser K. Hurt E. Nature. 2001; 413: 648-652Crossref PubMed Scopus (222) Google Scholar, 33Jensen T.H. Boulay J. Rosbash M. Libri D. Curr. Biol. 2001; 11: 1711-1715Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In our transcription elongation assays, sub2 confers a reduction in transcription elongation efficiency of 30-36% of the wild-type levels (Figs. 1 and 3). This proves clearly that Sub2 is also required for efficient transcription elongation in vitro. Examples of the stimulatory effects of proteins involved in mRNA metabolism, such as splicing proteins, on transcription elongation has been reported previously. Thus, in HeLa cell extracts, specific U1 and U2 small nuclear RNPs and Sm splicing proteins are loaded onto the transcription machinery via positive transcription elongation factor b (P-TEFb) interaction and are necessary for efficient transcription elongation (47Fong Y.W. Zhou Q. Nature. 2001; 414: 929-933Crossref PubMed Scopus (277) Google Scholar). As shown previously for mRNA export activity, the Sub2 requirement for transcription elongation is splicing-independent. This conclusion is based on two pieces of evidence. First, the elongation defect is observed in an intron-free system (double G-less cassette). Second, at permissive temperatures sub2-201 extracts are splicing-proficient (28Libri D. Graziani N. Saguez C. Boulay J. Genes Dev. 2001; 15: 36-41Crossref PubMed Scopus (111) Google Scholar), but transcription is elongation-deficient (Fig. 3). The observation that transcription elongation in heat-inactivated sub2-201 extracts is more severely impeded than in THO mutant extracts indicates that Sub2 might be a key element in facilitating transcription elongation. This supports the idea that Sub2-Yra1 and THO represent two distinct subcomplexes of TREX. It has been hypothesized that THO may be loaded onto RNAPII-mRNP structures prior to Sub2, helping to recruit it (22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodríguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (636) Google Scholar, 37Zenklusen D. Vinciguerra P. Wyss J.C. Stutz F. Mol. Cell. Biol. 2002; 22: 8241-8253Crossref PubMed Scopus (254) Google Scholar). This is in accordance with our in vitro results and the observations that multicopy sub2 suppresses both the transcription and recombination phenotypes of hpr1Δ cells (23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar, 24Fan H.Y. Merker R.J. Klein H.L. Mol. Cell. Biol. 2001; 21: 5459-5470Crossref PubMed Scopus (49) Google Scholar). The negative effect on transcription elongation of the THO/TREX mutations does not necessarily indicate that THO or Sub2-Yra1 are bona fide transcription elongation factors (48Aguilera A. EMBO J. 2002; 21: 195-201Crossref PubMed Scopus (264) Google Scholar). In this sense, spt4Δ mutants show similar in vitro and in vivo transcription elongation defects as do THO mutants. However, in contrast to THO mutants, in spt4Δ cells the in vitro transcription elongation defect is stronger; impairment of transcription does not lead to hyper-recombination, and there are no mRNA export defects (39Rondón A.G. García-Rubio M. González-Barrera S. Aguilera A. EMBO J. 2003; 22: 612-620Crossref PubMed Scopus (79) Google Scholar). The interconnection between the different steps of mRNA metabolism from transcription to mRNA processing and export (49Proudfoot N.J. Furger A. Dye M.J. Cell. 2002; 108: 501-512Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar) suggests the possibility that a set of proteins without a primary role in transcription may also affect transcription elongation. Consistent with this view, mutations in the Mex67·Mtr2 and Thp1·Sac3 complexes confer similar transcription and recombination phenotypes as the THO mutants do (23Jimeno S. Rondón A.G. Luna R. Aguilera A. EMBO J. 2002; 21: 3526-3535Crossref PubMed Scopus (211) Google Scholar, 25Gallardo M. Aguilera A. Genetics. 2001; 157: 79-89PubMed Google Scholar, 27Gallardo M. Luna R. Erdjument-Bromage H. Tempst P. Aguilera A. J. Biol. Chem. 2003; 278: 24225-24232Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The effect of THO and Sub2 on transcription elongation and mRNA metabolism and export suggests the possibility, already raised, that the nascent mRNA might have a role in transcription elongation efficiency and transcription-associated hyper-recombination (48Aguilera A. EMBO J. 2002; 21: 195-201Crossref PubMed Scopus (264) Google Scholar). In summary, this study shows that there is a set of conserved eukaryotic factors, like THO/TREX, that function at the interface between transcription and mRNA metabolism and that, regardless of their specific functional roles, are required for efficient transcription elongation. Deciphering the function of these factors will contribute to understanding the mechanisms of transcription elongation, how it is coupled with mRNA processing and export, and the mechanisms of transcription-associated genetic instability. We thank J. Q. Svejstrup, D. Libri, and M. Ptashne for yeast strain and plasmid gifts, R. Luna for critical reading of the manuscript, and D. Haun for style supervision." @default.
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• W1973607544 title "Molecular Evidence That the Eukaryotic THO/TREX Complex Is Required for Efficient Transcription Elongation" @default.
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