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Loss of anticodon wobble uridine modifications affects tRNA(Lys) function and protein levels in Saccharomyces cerevisiae.

Klassen R, Grunewald P, Thüring KL, Eichler C, Helm M, Schaffrath R - PLoS ONE (2015)

Bottom Line: Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect.Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU.These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.

ABSTRACT
In eukaryotes, wobble uridines in the anticodons of tRNA(Lys)UUU, tRNA(Glu)UUC and tRNA(Gln)UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

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Related in: MedlinePlus

Loss of mcm5s2 affects protein levels.(A) Analysis of cellular protein content of indicated strains. Identical numbers of cells were subjected to chemical lysis and analyzed by SDS-PAGE and Coomassie staining. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (B) Analysis of total protein extracts from indicated strains adjusted to identical protein concentration. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (C) Western detection of Pfk1 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (D) Western detection of Cdc19 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (E) Western detection of Pfk1 and Cdc19 in total protein extracts from indicated strains adjusted to identical protein concentration. Signal intensities relative to WT are indicated. (F) RT-PCR analysis of cDNA from indicated strains for PFK1, CDC19 and ACT1 mRNAs. Identical amounts of total RNA were subjected to reverse transcription.
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pone.0119261.g003: Loss of mcm5s2 affects protein levels.(A) Analysis of cellular protein content of indicated strains. Identical numbers of cells were subjected to chemical lysis and analyzed by SDS-PAGE and Coomassie staining. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (B) Analysis of total protein extracts from indicated strains adjusted to identical protein concentration. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (C) Western detection of Pfk1 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (D) Western detection of Cdc19 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (E) Western detection of Pfk1 and Cdc19 in total protein extracts from indicated strains adjusted to identical protein concentration. Signal intensities relative to WT are indicated. (F) RT-PCR analysis of cDNA from indicated strains for PFK1, CDC19 and ACT1 mRNAs. Identical amounts of total RNA were subjected to reverse transcription.

Mentions: To test whether the complete absence of mcm5s2U or other incorrect wobble uridine modification scenarios may lead to protein synthesis defects, we analyzed total cellular protein levels in strains with different modification defects (Fig. 3A). We used elp3 and uba4 single and double mutants as well as trm9 mutants carrying ncm5/ncm5s2U instead of mcm5/mcm5s2U [16]. For each strain, identical numbers of cells were subjected to chemical lysis and subsequently analyzed by SDS PAGE and Coomassie staining. While there were smaller effects in the trm9, uba4 and elp3 single mutants, total cellular protein content was markedly decreased in the elp3uba4 double mutant (Fig. 3A). We also monitored differences in protein content for Cdc19 (pyruvate kinase; [32]) and Pfk1 (phosphofructokinase; [33]) and found that in general, differences in abundance are comparable to the effects on total protein levels (Fig. 3C, 3D). Both proteins are already reduced in abundance in the elp3 single mutant. In the complete absence of mcm5s2 (elp3uba4), however, Cdc19 is hardly detectable, while Pfk1 becomes entirely undetectable, indicating a significant negative impact of the tRNA modification defect on the abundance of these two proteins. In comparison to elp3 and elp3uba4 strains, trm9 and uba4 single mutants less severely decreased the abundance of Pfk1. For Cdc19, levels were slightly decreased in trm9 but not in uba4 backgrounds (Fig. 3D). To verify equal cell numbers, portions of adjusted cell suspensions were removed before initializing chemical lysis, serially diluted and spotted on YPD. As shown in Fig. 3C and 3D, all tRNA modification mutants, including the elp3uba4 mutant, where Pfk1 and Cdc19 signals are severely reduced, formed comparable numbers of viable cells in individual spots, indicating a similar input to chemical lysis. Since the drop dilution method might not be suitable to detect smaller changes in viable cell titers, we grew wild type cells along with the elp3uba4 double mutant to early exponential phase (OD600nm ∼0.5), adjusted both cultures to OD600nm = 1 and determined exact total and viable cell titers by hemocytometry and viability plating. Indeed, there is a slight deviation (−13.9% for elp3uba4 compared to WT) in total cell numbers which is likely attributable to above mentioned changes in morphology that affect OD600nm/total cell number ratios (S2 Fig.). Moreover, we find that elp3uba4 double mutants exhibit an even greater loss of viable cell counts (−22.6% compared to WT), indicating a significant accumulation of dead cells. We confirmed this latter notion by staining dead cells with methylene blue. While these are essentially absent in the exponential WT culture, dead cells are detectable for the elp3uba4 strain. In addition, we observed a number of spontaneously lysed cells, which stain dark in phase contrast and weakly positive with methylene blue. Interestingly, dead staining and lysis appears to correlate with the elongated/multiple bud phenotype (S2 Fig.). Thus, tRNA hypomodification-induced cell death likely contributes to the observed differences in protein levels but the extent of viability loss (22.6%) appears to be insufficient to solely explain the observed differences in protein abundance. Semi-quantitative RT-PCR analysis of total mRNA excluded the possibility of a severe drop in transcription of CDC19, PFK1 or ACT1 genes in the elp3uba4 double mutant, strongly suggesting the significantly reduced abundance of Cdc19 and Pfk1 in this strain results from a translational rather than a transcriptional defect or a combination of a translational defect with the induction cell death (Fig. 3F).


Loss of anticodon wobble uridine modifications affects tRNA(Lys) function and protein levels in Saccharomyces cerevisiae.

Klassen R, Grunewald P, Thüring KL, Eichler C, Helm M, Schaffrath R - PLoS ONE (2015)

Loss of mcm5s2 affects protein levels.(A) Analysis of cellular protein content of indicated strains. Identical numbers of cells were subjected to chemical lysis and analyzed by SDS-PAGE and Coomassie staining. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (B) Analysis of total protein extracts from indicated strains adjusted to identical protein concentration. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (C) Western detection of Pfk1 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (D) Western detection of Cdc19 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (E) Western detection of Pfk1 and Cdc19 in total protein extracts from indicated strains adjusted to identical protein concentration. Signal intensities relative to WT are indicated. (F) RT-PCR analysis of cDNA from indicated strains for PFK1, CDC19 and ACT1 mRNAs. Identical amounts of total RNA were subjected to reverse transcription.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352028&req=5

pone.0119261.g003: Loss of mcm5s2 affects protein levels.(A) Analysis of cellular protein content of indicated strains. Identical numbers of cells were subjected to chemical lysis and analyzed by SDS-PAGE and Coomassie staining. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (B) Analysis of total protein extracts from indicated strains adjusted to identical protein concentration. The band marked with (*) was quantified and relative intensities compared to WT indicated below. (C) Western detection of Pfk1 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (D) Western detection of Cdc19 levels from indicated strains after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below. Before lysis, suspensions were serially diluted and spotted on YPD to confirm equal cell densities (input control). (E) Western detection of Pfk1 and Cdc19 in total protein extracts from indicated strains adjusted to identical protein concentration. Signal intensities relative to WT are indicated. (F) RT-PCR analysis of cDNA from indicated strains for PFK1, CDC19 and ACT1 mRNAs. Identical amounts of total RNA were subjected to reverse transcription.
Mentions: To test whether the complete absence of mcm5s2U or other incorrect wobble uridine modification scenarios may lead to protein synthesis defects, we analyzed total cellular protein levels in strains with different modification defects (Fig. 3A). We used elp3 and uba4 single and double mutants as well as trm9 mutants carrying ncm5/ncm5s2U instead of mcm5/mcm5s2U [16]. For each strain, identical numbers of cells were subjected to chemical lysis and subsequently analyzed by SDS PAGE and Coomassie staining. While there were smaller effects in the trm9, uba4 and elp3 single mutants, total cellular protein content was markedly decreased in the elp3uba4 double mutant (Fig. 3A). We also monitored differences in protein content for Cdc19 (pyruvate kinase; [32]) and Pfk1 (phosphofructokinase; [33]) and found that in general, differences in abundance are comparable to the effects on total protein levels (Fig. 3C, 3D). Both proteins are already reduced in abundance in the elp3 single mutant. In the complete absence of mcm5s2 (elp3uba4), however, Cdc19 is hardly detectable, while Pfk1 becomes entirely undetectable, indicating a significant negative impact of the tRNA modification defect on the abundance of these two proteins. In comparison to elp3 and elp3uba4 strains, trm9 and uba4 single mutants less severely decreased the abundance of Pfk1. For Cdc19, levels were slightly decreased in trm9 but not in uba4 backgrounds (Fig. 3D). To verify equal cell numbers, portions of adjusted cell suspensions were removed before initializing chemical lysis, serially diluted and spotted on YPD. As shown in Fig. 3C and 3D, all tRNA modification mutants, including the elp3uba4 mutant, where Pfk1 and Cdc19 signals are severely reduced, formed comparable numbers of viable cells in individual spots, indicating a similar input to chemical lysis. Since the drop dilution method might not be suitable to detect smaller changes in viable cell titers, we grew wild type cells along with the elp3uba4 double mutant to early exponential phase (OD600nm ∼0.5), adjusted both cultures to OD600nm = 1 and determined exact total and viable cell titers by hemocytometry and viability plating. Indeed, there is a slight deviation (−13.9% for elp3uba4 compared to WT) in total cell numbers which is likely attributable to above mentioned changes in morphology that affect OD600nm/total cell number ratios (S2 Fig.). Moreover, we find that elp3uba4 double mutants exhibit an even greater loss of viable cell counts (−22.6% compared to WT), indicating a significant accumulation of dead cells. We confirmed this latter notion by staining dead cells with methylene blue. While these are essentially absent in the exponential WT culture, dead cells are detectable for the elp3uba4 strain. In addition, we observed a number of spontaneously lysed cells, which stain dark in phase contrast and weakly positive with methylene blue. Interestingly, dead staining and lysis appears to correlate with the elongated/multiple bud phenotype (S2 Fig.). Thus, tRNA hypomodification-induced cell death likely contributes to the observed differences in protein levels but the extent of viability loss (22.6%) appears to be insufficient to solely explain the observed differences in protein abundance. Semi-quantitative RT-PCR analysis of total mRNA excluded the possibility of a severe drop in transcription of CDC19, PFK1 or ACT1 genes in the elp3uba4 double mutant, strongly suggesting the significantly reduced abundance of Cdc19 and Pfk1 in this strain results from a translational rather than a transcriptional defect or a combination of a translational defect with the induction cell death (Fig. 3F).

Bottom Line: Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect.Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU.These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.

ABSTRACT
In eukaryotes, wobble uridines in the anticodons of tRNA(Lys)UUU, tRNA(Glu)UUC and tRNA(Gln)UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

Show MeSH
Related in: MedlinePlus