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The exosome cofactor Rrp47 is critical for the stability and normal expression of its associated exoribonuclease Rrp6 in Saccharomyces cerevisiae.

Feigenbutz M, Garland W, Turner M, Mitchell P - PLoS ONE (2013)

Bottom Line: Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability.Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs.These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology and Biotechnology Department, The University of Sheffield, Sheffield, United Kingdom.

ABSTRACT
Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3' end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47. Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability. Exogenous expression of Rrp6 enables normal levels to be attained in rrp47∆ mutants. Strikingly, exogenous expression of Rrp6 suppresses many, but not all, of the RNA processing and maturation defects observed in an rrp47∆ mutant and complements the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs. In contrast, increased Rrp6 expression in the rrp47∆ mpp6∆ double mutant diminishes the block in the turnover of CUTs and in the degradation of the substrates of RNA discard pathways. These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs.

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Rrp6 protein stability is decreased in the rrp47∆ mutant.Isogenic wild-type and rrp47∆ strains were harvested during growth in selective minimal medium (SD) or rich medium (YPD) and at time-points after addition of the translation inhibitor cycloheximide (CHX), as indicated. Extracts were prepared under denaturing conditions and identical western blots were incubated with antiserum specific to Rrp6 and the loading control Pgk1. (A) Translational shut-off experiment in YPD medium. (B) Translational shut-off experiment in SD medium. (C) Quantitative analysis of the amount of Rrp6 in extracts from wild-type and rrp47∆ strains before addition of cycloheximide (“0” lanes) and 60 minutes after treatment (“60” lanes). The relative amount of Rrp6, normalised to Pgk1 expression levels and standardised to the level observed in the wild-type strain during growth in YPD medium (average of 2 experiments), is given below each lane.
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pone-0080752-g002: Rrp6 protein stability is decreased in the rrp47∆ mutant.Isogenic wild-type and rrp47∆ strains were harvested during growth in selective minimal medium (SD) or rich medium (YPD) and at time-points after addition of the translation inhibitor cycloheximide (CHX), as indicated. Extracts were prepared under denaturing conditions and identical western blots were incubated with antiserum specific to Rrp6 and the loading control Pgk1. (A) Translational shut-off experiment in YPD medium. (B) Translational shut-off experiment in SD medium. (C) Quantitative analysis of the amount of Rrp6 in extracts from wild-type and rrp47∆ strains before addition of cycloheximide (“0” lanes) and 60 minutes after treatment (“60” lanes). The relative amount of Rrp6, normalised to Pgk1 expression levels and standardised to the level observed in the wild-type strain during growth in YPD medium (average of 2 experiments), is given below each lane.

Mentions: The reduction in Rrp47 observed in an rrp6∆ mutant is principally due to a decrease in protein stability when Rrp6 is not available for interaction [43]. To determine whether Rrp6 is less stable in the absence of Rrp47, cultures of isogenic wild-type and rrp47∆ strains were treated with the translation inhibitor cycloheximide and the depletion of non-tagged, wild-type Rrp6 was followed by western analyses of cell extracts. Rrp6 levels showed a clear decrease through the 80 minute time-course in the rrp47∆ mutant, compared to the wild-type strain (Figure 2A,B). Quantitative analyses show that after 60 minutes incubation the Rrp6 levels were reduced by ~ 25 % in the wild-type strain, whereas the reduction was nearly 10-fold in the rrp47∆ mutant (Figure 2C). The half-life of the Rrp6 protein was estimated to be ~ 25 minutes in the rrp47∆ mutant and greater than 80 minutes in the wild-type strain (Figure S1). The stability of Rrp6 in the rrp47∆ mutant relative to the wild-type strain was not further decreased when the cultures were grown in minimal medium (Figures 2 and S1). These results show that Rrp6 is more rapidly degraded in the absence of Rrp47, and suggest that an additional mechanism is responsible for the exacerbated decrease in Rrp6 steady state levels during growth in minimal medium. Quantitative real time PCR (qPCR) analyses revealed that the expression level of RRP6 mRNA in the rrp47∆ mutant was reduced to ~ 60% of the level observed in wild-type cells during growth in minimal medium (62.5 %, SEM=3.6 %, n=4), while a slight increase in RRP6 mRNA levels was observed in the rrp47∆ mutant during growth in rich medium (114 %, SEM=8.7 %, n=4) (Figure 3). Taken together with the western blotting data, these results are consistent with a decrease in Rrp6 levels in the rrp47∆ mutant due to a general decrease in Rrp6 protein stability that is augmented by a decrease in RRP6 mRNA levels during growth in minimal medium.


The exosome cofactor Rrp47 is critical for the stability and normal expression of its associated exoribonuclease Rrp6 in Saccharomyces cerevisiae.

Feigenbutz M, Garland W, Turner M, Mitchell P - PLoS ONE (2013)

Rrp6 protein stability is decreased in the rrp47∆ mutant.Isogenic wild-type and rrp47∆ strains were harvested during growth in selective minimal medium (SD) or rich medium (YPD) and at time-points after addition of the translation inhibitor cycloheximide (CHX), as indicated. Extracts were prepared under denaturing conditions and identical western blots were incubated with antiserum specific to Rrp6 and the loading control Pgk1. (A) Translational shut-off experiment in YPD medium. (B) Translational shut-off experiment in SD medium. (C) Quantitative analysis of the amount of Rrp6 in extracts from wild-type and rrp47∆ strains before addition of cycloheximide (“0” lanes) and 60 minutes after treatment (“60” lanes). The relative amount of Rrp6, normalised to Pgk1 expression levels and standardised to the level observed in the wild-type strain during growth in YPD medium (average of 2 experiments), is given below each lane.
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Related In: Results  -  Collection

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pone-0080752-g002: Rrp6 protein stability is decreased in the rrp47∆ mutant.Isogenic wild-type and rrp47∆ strains were harvested during growth in selective minimal medium (SD) or rich medium (YPD) and at time-points after addition of the translation inhibitor cycloheximide (CHX), as indicated. Extracts were prepared under denaturing conditions and identical western blots were incubated with antiserum specific to Rrp6 and the loading control Pgk1. (A) Translational shut-off experiment in YPD medium. (B) Translational shut-off experiment in SD medium. (C) Quantitative analysis of the amount of Rrp6 in extracts from wild-type and rrp47∆ strains before addition of cycloheximide (“0” lanes) and 60 minutes after treatment (“60” lanes). The relative amount of Rrp6, normalised to Pgk1 expression levels and standardised to the level observed in the wild-type strain during growth in YPD medium (average of 2 experiments), is given below each lane.
Mentions: The reduction in Rrp47 observed in an rrp6∆ mutant is principally due to a decrease in protein stability when Rrp6 is not available for interaction [43]. To determine whether Rrp6 is less stable in the absence of Rrp47, cultures of isogenic wild-type and rrp47∆ strains were treated with the translation inhibitor cycloheximide and the depletion of non-tagged, wild-type Rrp6 was followed by western analyses of cell extracts. Rrp6 levels showed a clear decrease through the 80 minute time-course in the rrp47∆ mutant, compared to the wild-type strain (Figure 2A,B). Quantitative analyses show that after 60 minutes incubation the Rrp6 levels were reduced by ~ 25 % in the wild-type strain, whereas the reduction was nearly 10-fold in the rrp47∆ mutant (Figure 2C). The half-life of the Rrp6 protein was estimated to be ~ 25 minutes in the rrp47∆ mutant and greater than 80 minutes in the wild-type strain (Figure S1). The stability of Rrp6 in the rrp47∆ mutant relative to the wild-type strain was not further decreased when the cultures were grown in minimal medium (Figures 2 and S1). These results show that Rrp6 is more rapidly degraded in the absence of Rrp47, and suggest that an additional mechanism is responsible for the exacerbated decrease in Rrp6 steady state levels during growth in minimal medium. Quantitative real time PCR (qPCR) analyses revealed that the expression level of RRP6 mRNA in the rrp47∆ mutant was reduced to ~ 60% of the level observed in wild-type cells during growth in minimal medium (62.5 %, SEM=3.6 %, n=4), while a slight increase in RRP6 mRNA levels was observed in the rrp47∆ mutant during growth in rich medium (114 %, SEM=8.7 %, n=4) (Figure 3). Taken together with the western blotting data, these results are consistent with a decrease in Rrp6 levels in the rrp47∆ mutant due to a general decrease in Rrp6 protein stability that is augmented by a decrease in RRP6 mRNA levels during growth in minimal medium.

Bottom Line: Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability.Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs.These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology and Biotechnology Department, The University of Sheffield, Sheffield, United Kingdom.

ABSTRACT
Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3' end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47. Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability. Exogenous expression of Rrp6 enables normal levels to be attained in rrp47∆ mutants. Strikingly, exogenous expression of Rrp6 suppresses many, but not all, of the RNA processing and maturation defects observed in an rrp47∆ mutant and complements the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs. In contrast, increased Rrp6 expression in the rrp47∆ mpp6∆ double mutant diminishes the block in the turnover of CUTs and in the degradation of the substrates of RNA discard pathways. These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs.

Show MeSH
Related in: MedlinePlus