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. 2007 Aug 16:8:71.
doi: 10.1186/1471-2199-8-71.

Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae

Svetlana Chabelskaya  1 , Valentina Gryzina, Svetlana Moskalenko, Catherine Le Goff, Galina Zhouravleva
Affiliations

Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae

Svetlana Chabelskaya et al. BMC Mol Biol. .

Abstract

Background: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.

Results: In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Delta double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants.

Conclusion: This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.

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Figures

Figure 1
Figure 1
his7-1 mRNA accumulated when nonsense-mediated decay is inhibited. Nothern blotting was used to assess the effect of UPF1 deletion on the accumulation of his7-1 mRNA. Total RNA was isolated from strain 5B-D1645 (his7-1 upf1Δ) transformed with plasmids pRS316 and pRS316/UPF1, designated as (upf1Δ) and (UPF1), respectively. Northern blots were hybridized with radiolabeled HIS7, ADE1, ACT1 and CYH2 probes. A. Representative hybridization signals specific to his7-1 mRNA (upper panel), ade1-14 mRNA (middle panel) and actin mRNA (ACT1) used as an internal control (lower panel) are shown. Numbers indicated under upper and middle panels represent the relative abundance of his7-1 and ade1-14 mRNA's, respectively, in upf1Δ and UPF1 strains. (s.d.) – standard deviation. B. Accumulation of CYH2 precursor mRNA was used to control that NMD is altered in the upf1Δ strain. The CYH2 probe detects both precursor and mature CYH2 mRNA. The fold increase in CYH2 precursor/mature mRNA accumulation in upf1Δ strain relative to UPF1 strain is indicated with the standard deviation (s.d.).
Figure 2
Figure 2
Nonsense or missense alleles of SUP45 affect accumulation of his7-1 mRNA and CYH2 pre-mRNA. Northern blots were prepared with total RNA from wild-type strain 1B-D1606 (SUP45) and its sup45 mutant derivatives. Blots were hybridized with DNA probes that detected the his7-1, CYH2 and ACT1 transcripts. For each mutant the average CYH2 pre-mRNA/mRNA ratio (A) and the abundance of his7-1 mRNA (B) relative to the wild-type strain are shown with the standard deviation (s.d.). Following sup45 mutations were tested: 102, 104, 105, 107 (nonsense) and 103 (missense).
Figure 3
Figure 3
Deletion of UPF1 gene leads to increased viability of sup45 nonsense mutants. (A). Strains 1A-D1628 (sup45Δ) and 1-1A-D1628 (sup45Δ upf1Δ) all containing SUP45 deletion and pRS316/SUP45 plasmid were transformed with pRS315/SUP45-LEU2 plasmids carrying different sup45 mutant alleles. The growth of the transformants was tested by plating 100, and 10-1 serial dilutions of overnight cultures (left to right) onto 5-FOA plates. The extent of cell growth on 5-FOA plates indicates the ability of the sup45 mutant alleles to support cell growth in the presence and absence of UPF1 gene. The same serially diluted cultures were also spotted on synthetic complete plates lacking leucine and uracil (-L -U) to estimate the total number of cells analyzed. (B). Level of eRF1 protein in the clones selected on 5-FOA medium was analyzed by western blot. Tubulin was used as a loading control. Following sup45 mutations were tested: 102, 107 (nonsense) and 103 (missense).
Figure 4
Figure 4
Deletion of the UPF1 gene suppresses several sup45 phenotypes. The growth of the transformants of two isogenic strains, 1A-D1628 (sup45Δ) and 1-1A-D1628 (sup45Δ upf1Δ), selected on 5-FOA medium (Fig. 3A) was tested by plating 100, and 10-1 serial dilutions of overnight cultures (left to right) onto YPD plates at 37°C (A), onto YPD plates with 1 mg/ml paromomycin (B) and onto synthetic complete -adenine -leucine plates (C). The same serially diluted cultures were also spotted on YPD plates at 25°C (D) to estimate the total number of cells analyzed. Following sup45 mutations were tested: 102, 107 (nonsense) and 103 (missense).
Figure 5
Figure 5
Double mutants sup45 upf1Δ are characterized by defects of NMD. A. Representative hybridization signals specific to precursor and mature forms of CYH2. Total RNA was isolated from strain 3v-D1658 (sup45Δ upf1Δ pRS315/SUP45) and its derivates (sup45Δ upf1Δ pRS315/sup45-n) transformed with pRS316 and pRS316/UPF1 plasmids, designated as (UPF1 -) and (UPF1 +), respectively. The Northern blots were hybridized with radiolabeled CYH2 probe. The CYH2 precursor/mature ratio in wild-type strain was set as 1.0. B. The fold increase in CYH2 precursor/mature mRNA accumulation measured in the same strains as in panel A are represented relative to such in wild-type strain. C. The same transformants as in panel A were tested by plating 100, 10-1and 10-2 serial dilutions of overnight cultures (left to right) on synthetic complete plates lacking adenine and incubated 5 days at 25°C. The same serially diluted cultures were also spotted on plates lacking leucine and uracil (-L -U) to estimate the total number of cells analyzed. D. Norhern blots prepared with total RNA from the same transformants as in panel A were hybridized with radiolabeled probes, detecting ade1-14, SUP45 and scR1 mRNA (scR1 was used as a control). eRF1 and eRF3 protein levels in the same transformants were analyzed by western blot. Tubulin was used as a loading control. WT – wild type, 102 – sup45-102 (nonsense), 107 – sup45-107 (nonsense).
Figure 6
Figure 6
Deletion either UPF2 or UPF3 gene leads to increased viability of sup45 nonsense mutants. Strains 4a-D1659 (sup45Δ) and 4b-D1659 (sup45Δ upf2Δ) (A), 18a-D1660 (sup45Δ) and 3a-D1660 (sup45Δ upf3Δ) (B), all containing SUP45 deletion and pRS316/SUP45 plasmid were transformed with pRS315/sup45-n-LEU2 plasmids carrying different sup45 mutant alleles. The growth of the transformants was tested by plating 100, and 101 serial dilutions of overnight cultures (left to right) onto 5-FOA plates. The extent of cell growth on 5-FOA plates indicates the ability of the sup45 mutant alleles to support cell growth in the presence and absence of UPF2 or UPF3 genes. The same serially diluted cultures were also spotted on synthetic complete plates lacking leucine and uracil to estimate the total number of cells analyzed. (C, D) eRF1 and eRF3 protein levels in the same transformants as in panels A and B were analyzed by western blot. Tubulin was used as a loading control. Following sup45 mutations were tested: 102, 107 (nonsense) and 103 (missense).
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