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A genetic screen in Saccharomyces cerevisiae identifies new genes that interact with mex67-5, a temperature-sensitive allele of the gene encoding the mRNA export receptor

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Abstract

The Mex67p protein, together with Mtr2p, functions as the mRNA export receptor in Saccharomyces cerevisiae by interacting with both mRNA and nuclear pore complexes. To identify genes that interact functionally with MEX67, we used transposon insertion to search for mutations that suppressed the temperature-sensitive mex67-5 allele. Four suppressors are described here. The screen revealed that mutant Mex67-5p, but not wild-type Mex67p, is a target of the nuclear protein quality control mediated by San1p, a ubiquitin-protein ligase that participates in degradation of aberrant chromatin-associated proteins. Our finding that overexpression of the SPT6 gene alleviates the growth defects of the mex67-5 strain, together with the impairment of poly(A)+ RNA export caused by depletion of Spt6p or the related protein Iws1p/Spn1p, supports the mechanism proposed in mammalian cells for Spt6-mediated co-transcriptional loading of mRNA export factors during transcription elongation. Finally, our results also uncovered genetic connections between Mex67p and the poly(A) nuclease complex and with components of chromatin boundary elements.

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Acknowledgments

We thank S. Chávez, S. Rodríguez-Navarro and M. Pamblanco for providing plasmids and yeast strains. This work was supported by grants from the Spanish Ministry of Education and Science (BFU2004-00069/BMC and BFU2007-61525/BMC) to F.E and from the National Institutes of Health (GM33998) to C.N.C. L.P.-C. is recipient of a fellowship from the F.P.I program of the Spanish Ministry of Education and Science.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Universitat de Valencia, c/Dr. Moliner, 50, Burjassot (Valencia), 46100, Spain

    Francisco Estruch, Lorena Peiró-Chova, Natalia Gómez-Navarro, Jordi Durbán & Marceŀlí del Olmo

  2. Departments of Biochemistry and Genetics, Dartmouth Medical School, Dartmouth College, Hanover, NH, USA

    Christine Hodge & Charles N. Cole

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  1. Francisco Estruch
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Correspondence to Francisco Estruch.

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Communicated by M. Collart.

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438_2008_402_MOESM1_ESM.tif

Supplemental Figure 1. Deletion of the YTA7 gene does not have an effect on the nuclear accumulation of poly(A)+ RNA in mex67-5. Single mutant mex67-5 and double mutant mex67-5 Δyta7 strains were grown to mid-log phase at 30° and incubated for 1 hour at 33°. In all the cases, cells were fixed and in situ hybridization was performed using a Cy3-oligo(dT) probe [poly(A)]. DNA was visualized by DAPI staining. (TIFF 927 kb)

438_2008_402_MOESM2_ESM.tif

Supplemental Figure 2. Transposon insertion in the SPT6 promoter increases the Spt6p-protein level. Cells expressing a Spt6p-Myc fusion protein in the mex67-5 mutant or in the transposon-containing strain were grown at 30º. Total protein extracts were analyzed on 6% SDS-PAGE gel. Spt6p-13myc was detected with mouse monoclonal α-myc and anti-mouse immunoglobulin G-HRP as the secondary antibody. Ponceau staining of the Western blot is shown as a loading control. (TIFF 396 kb)

438_2008_402_MOESM3_ESM.tif

Supplemental Figure 3. Increased expression of the IWS1 gene in cells transformed with the pIWS1 plasmid. mex67-5 mutant cells were transformed with the pCM189 vector and with pIWS1, containing the IWS1 gene under the control of the tetO promoter. Total RNA was prepared from cells during exponential growth in selective medium. The gene encoding the ribosomal protein Rps23B is adjacent to IWS1, and RPS23B transcript was used as a loading control. (TIFF 282 kb)

438_2008_402_MOESM4_ESM.tif

Supplemental Figure 4. Overexpression of the SPT6 and IWS1 genes does not have an effect on the nuclear accumulation of poly(A)+ RNA in mex67-5. mex67-5 cells were transformed with plasmids expressing Spt6p (pSPT6) and Iws1p (pIWS1) under the control of the strong tetO promoter, or with the empty vector (pRS316). Strains were grown in selective medium to mid-log phase at 30° and incubated for 1 hour at 33°. In all cases, cells were fixed and in situ hybridization was performed using a Cy3-oligo(dT) probe [poly(A)]. DNA was visualized by DAPI staining (TIFF 1073 kb)

438_2008_402_MOESM5_ESM.tif

Supplemental Figure 5. Depletion of Spt6p and Iws1p by incubation of P GAL10 -SPT6 and P GAL10 -IWS1 strains in dextrose. Total protein extracts obtained from cells growing exponentially in YPgalactose or transferred to YPD for the indicated times were analyzed on 7% SDS-PAGE gel. Spt6p-HA and Iws1p-HA were detected by using mouse monoclonal antibody raised against the HA antigen. Loading of equal amounts of total protein was confirmed by Ponceau staining of the blot (not shown). (TIFF 600 kb)

438_2008_402_MOESM6_ESM.tif

Supplemental Figure 6. Depletion of the essential ER-localized protein Rot1p has only a minor effect on nuclear accumulation of poly(A)+. The indicated strains were grown at 30 ºC in YPGalactose to early exponential phase, diluted to an OD600 of 0.1 in YPD and incubated overnight. The black arrows indicate the slight nuclear staining observed in some cells depleted of Rot1p. White arrows indicate the strong nuclear accumulation observed in some cells depleted of Iws1p or Spt6p (see also Figure 7). In all cases, cells were fixed and in situ hybridization was performed using a Cy3-oligo(dT) probe to detect polyadenylated RNA. DNA was visualized by DAPI staining. (TIFF 1011 kb)

438_2008_402_MOESM7_ESM.tif

Supplemental Figure 7. Deletion of PAN2 or PAN3 does not reduce the hyperpolyadenylation shown by mex67-5 mutant. Poly(A) tail length determined according to Minvielle-Sebastia et al. (1998) by isolating total RNA from each strain that had been grown continuously at 30°C and shifted to 37°C for 60 min. The resulting RNAs were digested to completion with RNases T1 and A. The remaining oligo(A) and poly(A) fragments were then end labeled with [32P]pCp and RNA ligase and resolved on 9% polyacrylamide-7 M urea-TBE gels. (TIFF 2150 kb)

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Estruch, F., Peiró-Chova, L., Gómez-Navarro, N. et al. A genetic screen in Saccharomyces cerevisiae identifies new genes that interact with mex67-5, a temperature-sensitive allele of the gene encoding the mRNA export receptor. Mol Genet Genomics 281, 125–134 (2009). https://doi.org/10.1007/s00438-008-0402-x

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