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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 levels are not altered in mpp6∆ or rex1∆ mutants.Western analyses were performed on extracts from strains that either carry a wild-type or a deletion allele of the MPP6 or REX1 gene and that express the zz-Rrp6 fusion protein. Blots were successively incubated with the PAP antibody and antibody specific to the Pgk1 protein. Expression levels of zz-Rrp6 in the mpp6∆ and rex1∆ mutants, relative to the level observed in the corresponding wild-type strain, are given at the bottom of the figure and are the average of three independent biological replicates.
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pone-0080752-g009: Rrp6 levels are not altered in mpp6∆ or rex1∆ mutants.Western analyses were performed on extracts from strains that either carry a wild-type or a deletion allele of the MPP6 or REX1 gene and that express the zz-Rrp6 fusion protein. Blots were successively incubated with the PAP antibody and antibody specific to the Pgk1 protein. Expression levels of zz-Rrp6 in the mpp6∆ and rex1∆ mutants, relative to the level observed in the corresponding wild-type strain, are given at the bottom of the figure and are the average of three independent biological replicates.

Mentions: Given that the exonuclease activity of Rrp6 is redundant with activities dependent upon either Mpp6 or Rex1 [44](Figure 6), we hypothesised that the expression of Rrp6 may be increased when Mpp6- or Rex1-dependent pathways are blocked. To address this, we determined the relative levels of Rrp6 in extracts of isogenic strains that carry either a wild-type or allele of the MPP6 or REX1 gene. Western analyses of cultures grown in minimal medium showed that Rrp6 expression levels are not markedly altered in the presence or absence of Mpp6 or Rex1 (Figure 9). Thus, Rrp6 expression levels are responsive to the availability of its interacting protein Rrp47 but not the status of redundant Mpp6- or Rex1-dependent processing or degradation pathways.


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 levels are not altered in mpp6∆ or rex1∆ mutants.Western analyses were performed on extracts from strains that either carry a wild-type or a deletion allele of the MPP6 or REX1 gene and that express the zz-Rrp6 fusion protein. Blots were successively incubated with the PAP antibody and antibody specific to the Pgk1 protein. Expression levels of zz-Rrp6 in the mpp6∆ and rex1∆ mutants, relative to the level observed in the corresponding wild-type strain, are given at the bottom of the figure and are the average of three independent biological replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818262&req=5

pone-0080752-g009: Rrp6 levels are not altered in mpp6∆ or rex1∆ mutants.Western analyses were performed on extracts from strains that either carry a wild-type or a deletion allele of the MPP6 or REX1 gene and that express the zz-Rrp6 fusion protein. Blots were successively incubated with the PAP antibody and antibody specific to the Pgk1 protein. Expression levels of zz-Rrp6 in the mpp6∆ and rex1∆ mutants, relative to the level observed in the corresponding wild-type strain, are given at the bottom of the figure and are the average of three independent biological replicates.
Mentions: Given that the exonuclease activity of Rrp6 is redundant with activities dependent upon either Mpp6 or Rex1 [44](Figure 6), we hypothesised that the expression of Rrp6 may be increased when Mpp6- or Rex1-dependent pathways are blocked. To address this, we determined the relative levels of Rrp6 in extracts of isogenic strains that carry either a wild-type or allele of the MPP6 or REX1 gene. Western analyses of cultures grown in minimal medium showed that Rrp6 expression levels are not markedly altered in the presence or absence of Mpp6 or Rex1 (Figure 9). Thus, Rrp6 expression levels are responsive to the availability of its interacting protein Rrp47 but not the status of redundant Mpp6- or Rex1-dependent processing or degradation pathways.

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