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. 2021 Aug 15;22(16):8753.
doi: 10.3390/ijms22168753.

Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation

Bahar Khonsari  1 , Roland Klassen  1 , Raffael Schaffrath  1
Affiliations

Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation

Bahar Khonsari et al. Int J Mol Sci. .

Abstract

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.

Keywords: SSD1; pseudouridine; tRNA modification; yeast.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Temperature sensitivity of elp3 and deg1 mutants in the SSD1-v and ssd1-d strain backgrounds. (A) Wild type (WT) and indicated mutants were serially diluted, spotted on yeast extract–peptone–dextrose (YPD) plates, and incubated at the specified temperature for 48 h. (B) The WT and deg1 mutant in the ssd1-d background containing an empty vector, SSD1-v-, or ssd1-d-containing plasmids were serially diluted, spotted on YPD plates, and incubated at the indicated temperature for 48 h.
Figure 2
Figure 2
Comparison of the rapamycin sensitivity of tRNA modification mutants in SSD1-v and ssd1-d backgrounds. WT, elp3, and deg1 mutants of both background strains were serially diluted and spotted on YPD plates containing the indicated amounts of rapamycin. Plates were incubated at 30 °C for 48 h.
Figure 3
Figure 3
Plasmid shuffle assay to determine genetic interaction between DEG1 and ELP3 or URM1 in the ssd1-d strain. (A) Scheme indicating position and required genes for mcm5s2U34 and Ψ38/39 modifications in tRNA. (B) Principle of plasmid shuffle assay involving elp3 deg1 or urm1 deg1 double mutants carrying URA3-CEN plasmids that provide for ELP3 or URM1 wild-type gene functions, respectively. 5-FOA medium (FOA) counterselects against the URA3-based plasmids and thus uncovers the double mutant phenotype. (C) Result of plasmid shuffle assay in the deg1 urm1 strain. (D) Result of plasmid shuffle assay in the deg1 elp3 strain. WT and indicated mutants with and without URA3-based plasmids were serially diluted and spotted on YPD, URA, and FOA plates. YPD and URA plates were incubated for 48 h, and FOA plates were incubated for 72 h at 30 °C.
Figure 4
Figure 4
Comparison of protein levels of the glutamine-rich Rnq1-GFP fusion protein in absence of Ψ38/39 in SSD1-v and ssd1-d backgrounds. The total protein extract from indicated strains expressing Rnq1-GFP was used for Western analysis with anti-GFP and anti-Cdc19 antibodies. GFP and Cdc19 signal intensities were normalized to the respective WT intensity.
Figure 5
Figure 5
Impact of deg1 and ssd1-d on protein aggregation. Total protein and aggregate contents were extracted from (A) WT and deg1 mutants in both SSD1-v and ssd1-d backgrounds. (B) Total protein and aggregate contents from ssd1-d WT and the ssd1-d deg1 mutant in the presence (+) and absence (−) of plasmid-based SSD1-v [SSD1-v]. Samples were analyzed by Nu-PAGE and Coomassie staining.
Figure 6
Figure 6
Influence of SSD1 and DEG1 on chronological aging. (A) Chronological aging was analyzed for the SSD1-v WT and the ssd1-v deg1 mutant in comparison to the ssd1-d and ssd1-d deg1 strains over a time range of 17 days. Viability (%) represents the determined colony forming units (CFU) per ml normalized to the respective value at day 0. The mean of three independent cultures and the standard deviation is given. (B) As in (A) but with indicated strains in the presence or absence of plasmid-based SSD1-v [SSD1-v].
Figure 7
Figure 7
Comparison of the temperature sensitivity of other tRNA modification mutants in SSD1-v and ssd1-d backgrounds. (A) Wild-type (WT) and pus1, trm1, and ncl1 mutants in both SSD1-v and ssd1-d backgrounds were serially diluted, spotted on YPD plates, and incubated at elevated temperatures for 48 h. (B) As in (A) but using WT and trm8, ncl1, and trm8 ncl1 double mutants in both strain backgrounds.
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