The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability

Abstract

The THO complex is a multimeric factor containing four polypeptides, Tho2, Hpr1, Mft1 and Thp2. Mutations in any of the genes encoding THO confer impairment of transcription and a transcription-dependent hyper-recombination phenotype, suggesting that THO has a functional role in gene expression. Using an in vivo assay developed to study expression of long and G+C-rich DNA sequences, we have isolated SUB2, a gene involved in mRNA splicing and export, as a multicopy suppressor of the gene expression defect of hpr1 Delta. Further investigation of a putative functional relationship between mRNA metabolism and THO revealed that mRNA export mutants sub2, yra1, mex67 and mtr2 have similar defective transcription and hyper-recombination phenotypes as THO mutants. In addition, THO becomes essential in cells with a defective Mex67 mRNA export er. Finally, we have shown that THO has the ability to associate with RNA and DNA in vitro. These results indicate a functional link between the processes of elongation and metabolism of nascent mRNA mediated by THO and mRNA export proteins, which have important consequences for the maintenance of genome stability.

Fig. 1. Suppression of the transcriptional effect of hpr1 by overexpression of SUB2. (A) Scheme of the lacZ–URA3 fusion fragment under the control of the tet promoter used. (B) Phenotypic analysis of wild-type (W303-1A) and hpr1Δ (U678-1C) strains, transformed with either vector YEp351 or plasmids YEp351-SUB2 and YEp351-THO2 containing SUB2 and THO2, respectively. The capacity of each strain to form colonies on synthetic medium lacking uracil after 4 days at 30°C is shown on the top. On the bottom is the capacity of each strain to express β-galactosidase from plasmid pCM184-LAUR on complete synthetic medium, as determined by a colour assay. (C) Northern analysis of the expression of the Ptet::lacZ–URA3 fusion construct. RNA was isolated from mid-log phase cells carrying pCM184-LAUR grown in synthetic medium. As ³²P-labelled DNA probe we used a 3 kb BamHI lacZ fragment and the internal 589 bp 28S rRNA fragment obtained by PCR. RNA levels in arbitrary units (A.U.) were obtained in a Fuji FLA 3000 and were normalized with respect to rRNA levels of each sample. (D) Phenotypic analysis of hpr1Δ (U768-1C) cells transformed with Yep351-YRA1, YEp13-MEX67 and YEp13-MTR2.

Fig. 2. Genetic and molecular analysis of transcription in sub2Δ strains. (A) Phenotypic analysis of wild-type (CEN.PK) and sub2Δ isogenic strains. The capacity of each strain to form colonies in synthetic medium lacking uracil after 4 days at 30°C is shown on the top. On the bottom is the capacity of each strain to express β-galactosidase from plasmid pCM184-LAUR on complete synthetic medium, as determined by a colour assay. (B) Northern analysis of the expression of the Ptet::lacZ–URA3 and Ptet::LEU2 fusion constructs in yeast transformants carrying plasmids pCM184-LAUR and pCM189-LEU2, respectively. As ³²P-labelled LEU2 probe we used the ClaI–EcoRV LEU2 internal fragment. Other details as in Figure 1.

Fig. 3. Silver-stained SDS–PAGE of Tho2-TAP-tagged THO complex purified from wild-type and sub2Δ cells. The novel band (*) co-purifying with the TAP-tagged THO complex corresponds to Tex1 (Strässer et al., 2002).

Fig. 4. Expression analysis of GAL1pr::lacZ and GAL1pr::PHO5 constructs in wild-type (RS453a) and yra1-1 strains. (A) β-galactosidase and acid phosphatase activities of strains transformed with plasmids p416-GAL1–lacZ and pSCh202. The percentage value of activity is shown with respect to wild type (100%). Each value represents the average of two to three different transformants. Only data of induced expression are given (2% galactose). Under repressed conditions (2% glucose) values were below detection levels in all cases. (B) Northern analyses of lacZ and PHO5 mRNAs driven from the GAL1 promoter. Mid-log phase cells were diluted in 3% glycerol–2%lactate SC-ura fresh media to an OD₆₀₀ of 0.5 and incubated for 16 h. Galactose was then added and samples were taken at different times, as specified. As DNA probes we used the 0.9 kb EcoRV internal fragment of PHO5. For the lacZ and rRNA probes and other details see Figure 1.

Fig. 5. Expression analysis of GAL1pr::lacZ and GAL1pr::PHO5 constructs in mex67-5 (WMC1-1A), mtr2-ts26 and their isogenic wild-type strains (W303-1A and RS5453a, respectively). The experiments were carried out either at 30°C or after shifting cells to 37°C 30 min before activating transcription in media containing galactose. Details as in Figure 4.

Fig. 6. Genetic analysis of synthetic lethality of mex67-5 in combination with mutations of the THO complex. Tetrad analysis of heterozygous diploid strains obtained by crossing mex67-5 strains with either tho2Δ, hpr1Δ, mft1Δ or thp2Δ null mutants is shown (top). In hpr1Δ and tho2Δ the double mutant combinations were unable to germinate. In contrast to the single mutant mex67-5, the double mutant mex67-5 tho2Δ strain WMT2 transformed with plasmid pRS316THO2 containing a wild-type copy of THO2 was unable to form colonies on SC + 500 µg/ml of FOA (bottom).

Fig. 7. Recombination analyses of mutants affected in RNA metabolism. (A) Recombination frequencies of wild-type (RS453a) and yra1-1 and mex67-5 mutant strains transformed with plasmids pRS316LΔNS and pRS316L. The scheme of each system is shown. Arrows indicate the transcripts driven from the LEU2 promoter. For fluctuation tests, independent colonies were obtained from SC-ura and recombinants were selected in SC-leu-ura. (B) Recombination analysis of wild-type (MFM67-8A), mex67-5 (WML-6D), mft1Δ (MFM67-13A) and mex67 mft1Δ (MFM67-12C) strains using the chromosomal direct-repeat assay leu2-k::URA3-ADE2::leu2-k. (C) Recombination analysis of wild-type (MHJ130), hpr1Δ (U768-1C) and prp17Δ (MHJ131L) using the LI and LIr direct-repeat recombination assays containing the ACT1 intron between the 600 bp leu2 repeats in both the processed and unprocessed orientations, respectively. For fluctuation tests, independent colonies were obtained from SC and recombinants were selected in SC+FOA or SC-leu. The median frequency of six to 12 independent cultures is given in each case. All experiments were performed at 30°C.

Fig. 8. In vitro RNA and DNA binding activities of the His-tagged Tho2 purified THO complex. (A) RNA binding assay using the 90 bp ³²P-labelled RNA obtained from the pBluescript SK polylinker and increasing amounts of a preparation of purified complex. (B) Competition assays of the RNA-binding activity of the THO complex using as competitor increasing amounts of the same 90mer RNA, a 60mer ssDNA and a 200 bp dsDNA. (C) DNA binding assay using a 200 bp 5′-end ³²P-labelled, PCR-amplified dsDNA and increasing amounts of the same purified THO complex used in (A).

Fig. 9. Hypothetical scheme of the temporary scale of action of the THO complex and RNA export factors in relation to transcription. A question mark is shown at each step to denote our ignorance of the structural and temporary interaction between the different factors. The proteins could act on the nascent RNA sequentially as protein complexes. As a consequence of the link between transcription and RNA metabolism, a functional failure in any of these putative complexes could lead to both a defect in transcription and RNA export, regardless on their specific function. Different alternatives on how a transcriptional impairment can lead to hyper-recombination have been discussed elsewhere (see Aguilera, 2002).