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Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly.

Cary GA, Vinh DB, May P, Kuestner R, Dudley AM - G3 (Bethesda) (2015)

Bottom Line: Many of these proteins have been shown to form foci in response to other stresses.Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins.Thus, global characterization of PB composition has uncovered proteins important for PB assembly and evidence suggesting an active role for RNA in PB function.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, Washington 98109 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195.

No MeSH data available.


Related in: MedlinePlus

Analysis of RNA isolated in Dhh1−GFP complexes. (A) Representative microarray data from a mock immunoprecipitation (IP; green fluorescent protein alone) as well as Dhh1−GFP immunoisolations from a +glucose and a −glucose condition. The x-axis in each plot is the log-transformed, normalized array intensity for input (total) RNA, and the y-axis is the log-transformed, normalized array intensity for IP RNA. In each plot, the red line is a linear regression between the signals from the IP and total RNA, and the blue lines correspond to the 95% confidence interval. Transcripts above 95% confidence interval are considered enriched (and shaded black). Specific transcripts that are enriched and discussed in the text are highlighted and labeled (red, RPM1; brown, mitochondrial introns; purple, PAT1 and DCP2). (B) The proportion of each RNA class within the total identified 79 transcripts. ORF, open reading frame; SUT, stable unannotated transcript; CUT, cryptic unannotated transcript; MUT, meiotic unannotated transcript.
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fig4: Analysis of RNA isolated in Dhh1−GFP complexes. (A) Representative microarray data from a mock immunoprecipitation (IP; green fluorescent protein alone) as well as Dhh1−GFP immunoisolations from a +glucose and a −glucose condition. The x-axis in each plot is the log-transformed, normalized array intensity for input (total) RNA, and the y-axis is the log-transformed, normalized array intensity for IP RNA. In each plot, the red line is a linear regression between the signals from the IP and total RNA, and the blue lines correspond to the 95% confidence interval. Transcripts above 95% confidence interval are considered enriched (and shaded black). Specific transcripts that are enriched and discussed in the text are highlighted and labeled (red, RPM1; brown, mitochondrial introns; purple, PAT1 and DCP2). (B) The proportion of each RNA class within the total identified 79 transcripts. ORF, open reading frame; SUT, stable unannotated transcript; CUT, cryptic unannotated transcript; MUT, meiotic unannotated transcript.

Mentions: Because PB are known to depend on the presence of RNA for their integrity (Teixeira et al. 2005), we isolated RNA in parallel from each Dhh1–GFP immunopurified samples analyzed by MS. To facilitate the quantification of strand-specific transcripts in a manner that was independent on their polyadenylation status and not biased by the amplification protocol, we hybridized RNA directly to a custom DNA microarray and measured RNA abundance by using an antibody specific for RNA:DNA hybrids (Hu et al. 2006; Dutrow et al. 2008). Transcripts were considered enriched in the IP if they were above the 95% confidence interval of a linear regression between the normalized signal from matched IP and total RNA samples (Figure 4A). Using this method, we identified 79 transcripts that copurified with Dhh1–GFP in more than one biological replicate but not in the mock sample which immunoprecipitated GFP alone (Table S8). The majority of these 79 transcripts are significantly enriched for noncoding RNAs, including dubious ORF transcripts, cryptic unannotated transcript, stable unannotated transcript (Xu et al. 2009), and meiotic unannotated transcript (Lardenois et al. 2011) (Figure 4B). We also detect evidence of enrichment of Ty retroelements, along with proteins encoded by Ty elements, consistent with Ty association with PB (Checkley et al. 2010). Although the majority of identified transcripts are noncoding, we did not detect coenrichment of related transcripts, e.g., pairs of sense and antisense transcripts, which would suggest a regulatory role.


Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly.

Cary GA, Vinh DB, May P, Kuestner R, Dudley AM - G3 (Bethesda) (2015)

Analysis of RNA isolated in Dhh1−GFP complexes. (A) Representative microarray data from a mock immunoprecipitation (IP; green fluorescent protein alone) as well as Dhh1−GFP immunoisolations from a +glucose and a −glucose condition. The x-axis in each plot is the log-transformed, normalized array intensity for input (total) RNA, and the y-axis is the log-transformed, normalized array intensity for IP RNA. In each plot, the red line is a linear regression between the signals from the IP and total RNA, and the blue lines correspond to the 95% confidence interval. Transcripts above 95% confidence interval are considered enriched (and shaded black). Specific transcripts that are enriched and discussed in the text are highlighted and labeled (red, RPM1; brown, mitochondrial introns; purple, PAT1 and DCP2). (B) The proportion of each RNA class within the total identified 79 transcripts. ORF, open reading frame; SUT, stable unannotated transcript; CUT, cryptic unannotated transcript; MUT, meiotic unannotated transcript.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4632068&req=5

fig4: Analysis of RNA isolated in Dhh1−GFP complexes. (A) Representative microarray data from a mock immunoprecipitation (IP; green fluorescent protein alone) as well as Dhh1−GFP immunoisolations from a +glucose and a −glucose condition. The x-axis in each plot is the log-transformed, normalized array intensity for input (total) RNA, and the y-axis is the log-transformed, normalized array intensity for IP RNA. In each plot, the red line is a linear regression between the signals from the IP and total RNA, and the blue lines correspond to the 95% confidence interval. Transcripts above 95% confidence interval are considered enriched (and shaded black). Specific transcripts that are enriched and discussed in the text are highlighted and labeled (red, RPM1; brown, mitochondrial introns; purple, PAT1 and DCP2). (B) The proportion of each RNA class within the total identified 79 transcripts. ORF, open reading frame; SUT, stable unannotated transcript; CUT, cryptic unannotated transcript; MUT, meiotic unannotated transcript.
Mentions: Because PB are known to depend on the presence of RNA for their integrity (Teixeira et al. 2005), we isolated RNA in parallel from each Dhh1–GFP immunopurified samples analyzed by MS. To facilitate the quantification of strand-specific transcripts in a manner that was independent on their polyadenylation status and not biased by the amplification protocol, we hybridized RNA directly to a custom DNA microarray and measured RNA abundance by using an antibody specific for RNA:DNA hybrids (Hu et al. 2006; Dutrow et al. 2008). Transcripts were considered enriched in the IP if they were above the 95% confidence interval of a linear regression between the normalized signal from matched IP and total RNA samples (Figure 4A). Using this method, we identified 79 transcripts that copurified with Dhh1–GFP in more than one biological replicate but not in the mock sample which immunoprecipitated GFP alone (Table S8). The majority of these 79 transcripts are significantly enriched for noncoding RNAs, including dubious ORF transcripts, cryptic unannotated transcript, stable unannotated transcript (Xu et al. 2009), and meiotic unannotated transcript (Lardenois et al. 2011) (Figure 4B). We also detect evidence of enrichment of Ty retroelements, along with proteins encoded by Ty elements, consistent with Ty association with PB (Checkley et al. 2010). Although the majority of identified transcripts are noncoding, we did not detect coenrichment of related transcripts, e.g., pairs of sense and antisense transcripts, which would suggest a regulatory role.

Bottom Line: Many of these proteins have been shown to form foci in response to other stresses.Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins.Thus, global characterization of PB composition has uncovered proteins important for PB assembly and evidence suggesting an active role for RNA in PB function.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, Washington 98109 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195.

No MeSH data available.


Related in: MedlinePlus