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The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription.

Mirón-García MC, Garrido-Godino AI, Martínez-Fernández V, Fernández-Pevida A, Cuevas-Bermúdez A, Martín-Expósito M, Chávez S, de la Cruz J, Navarro F - Nucleic Acids Res. (2014)

Bottom Line: In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation.We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II.Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén, Paraje de las Lagunillas, s/n, 23071, Jaén, Spain.

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Lack of Bud27 affects the association between the RNA pol II and RSC chromatin remodeling complex. (A) The RNA pol II was immunoprecipitated with anti-Rpb1 antibody (8WG16), and Rpb1, Rpb1-CTDSer5P, Rpb1-CTDSer2P and Sth1-Myc were analysed by western blot with the antibodies indicated above in Sth1-Myc containing strains growing at 30ºC (left panel) in YPD medium. Ratios between Sth1 and Rpb1, Rpb1-CTDSer5P (Ser5) and Rpb1-CTDSer2P (Ser2) were calculated from the experiment in left panel (right panel). (B) Ratios between Rpb1-CTDSer5P (Ser5) or between Rpb1-CTDSer2P (Ser2) and Rpb1 were calculated from the experiment in A (left panel). Intensities of immunoreactive bands on western blots were quantified by densitometry using the software TOTALLAB from images acquired with office scanner. The data are the results of at least three different experiments.
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Figure 6: Lack of Bud27 affects the association between the RNA pol II and RSC chromatin remodeling complex. (A) The RNA pol II was immunoprecipitated with anti-Rpb1 antibody (8WG16), and Rpb1, Rpb1-CTDSer5P, Rpb1-CTDSer2P and Sth1-Myc were analysed by western blot with the antibodies indicated above in Sth1-Myc containing strains growing at 30ºC (left panel) in YPD medium. Ratios between Sth1 and Rpb1, Rpb1-CTDSer5P (Ser5) and Rpb1-CTDSer2P (Ser2) were calculated from the experiment in left panel (right panel). (B) Ratios between Rpb1-CTDSer5P (Ser5) or between Rpb1-CTDSer2P (Ser2) and Rpb1 were calculated from the experiment in A (left panel). Intensities of immunoreactive bands on western blots were quantified by densitometry using the software TOTALLAB from images acquired with office scanner. The data are the results of at least three different experiments.

Mentions: Based on previously reported data showing a preferential association of RSC with promoter regions versus ORF sequences and the difficulty of analysing its association with ORFs (18,38), we investigated the association between the Sth1 subunit of the RSC complex and RNA Pol II by performing protein co-immunoprecipitation (Figure 6). For this, we used both a wild-type and a bud27Δ mutant strain that expressed a functional Myc-tagged version of Sth1. To immunoprecipitate RNA pol II, we used 8WG16 antibodies raised against the CTD repeats of Rpb1, the largest subunit of this RNA polymerase. We found a very significant decrease in the amount of Sth1-Myc that co-immunoprecipitated with Rpb1. In addition, as the deletion of BUD27 leads to Rpb1 cytoplasmic accumulation (3), we analysed the levels of two phosphorylated forms of RNA pol II (CTD Ser5P and CTD Ser2P), accounting for elongating RNA pol II, in the immunoprecipitated preparations. Notably, these levels were not lower in bud27Δ than in the wild-type strain, and the ratios Sth1-Myc:CTD Ser2P and Sth1-Myc:CTD Ser5P in the bud27Δ mutant were as low as the Sth1-Myc:Rpb1 values (Figure 6A). These results were corroborated by performing similar analyses in chromatin fractions of the same strains (Supplementary Figure S4A). In addition, a sharp increase, at least in the Ser5P:Rpb1 ratio was found in bud27Δ with respect to a wild-type strain (Figure 6B), supporting a role by Bud27 during early elongation. Again, these data were corroborated in chromatin fractions (Supplementary Figure S4B). We conclude that Bud27 is necessary to maintain a correct association of RSC to RNA pol II during transcription. Overall, our results show a functional contribution of Bud27 to RNA pol II-dependent transcription that may be mediated by its role in promoting the interaction between the RSC complex and Rpb5, in the context of the elongating RNA polymerase.


The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription.

Mirón-García MC, Garrido-Godino AI, Martínez-Fernández V, Fernández-Pevida A, Cuevas-Bermúdez A, Martín-Expósito M, Chávez S, de la Cruz J, Navarro F - Nucleic Acids Res. (2014)

Lack of Bud27 affects the association between the RNA pol II and RSC chromatin remodeling complex. (A) The RNA pol II was immunoprecipitated with anti-Rpb1 antibody (8WG16), and Rpb1, Rpb1-CTDSer5P, Rpb1-CTDSer2P and Sth1-Myc were analysed by western blot with the antibodies indicated above in Sth1-Myc containing strains growing at 30ºC (left panel) in YPD medium. Ratios between Sth1 and Rpb1, Rpb1-CTDSer5P (Ser5) and Rpb1-CTDSer2P (Ser2) were calculated from the experiment in left panel (right panel). (B) Ratios between Rpb1-CTDSer5P (Ser5) or between Rpb1-CTDSer2P (Ser2) and Rpb1 were calculated from the experiment in A (left panel). Intensities of immunoreactive bands on western blots were quantified by densitometry using the software TOTALLAB from images acquired with office scanner. The data are the results of at least three different experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150788&req=5

Figure 6: Lack of Bud27 affects the association between the RNA pol II and RSC chromatin remodeling complex. (A) The RNA pol II was immunoprecipitated with anti-Rpb1 antibody (8WG16), and Rpb1, Rpb1-CTDSer5P, Rpb1-CTDSer2P and Sth1-Myc were analysed by western blot with the antibodies indicated above in Sth1-Myc containing strains growing at 30ºC (left panel) in YPD medium. Ratios between Sth1 and Rpb1, Rpb1-CTDSer5P (Ser5) and Rpb1-CTDSer2P (Ser2) were calculated from the experiment in left panel (right panel). (B) Ratios between Rpb1-CTDSer5P (Ser5) or between Rpb1-CTDSer2P (Ser2) and Rpb1 were calculated from the experiment in A (left panel). Intensities of immunoreactive bands on western blots were quantified by densitometry using the software TOTALLAB from images acquired with office scanner. The data are the results of at least three different experiments.
Mentions: Based on previously reported data showing a preferential association of RSC with promoter regions versus ORF sequences and the difficulty of analysing its association with ORFs (18,38), we investigated the association between the Sth1 subunit of the RSC complex and RNA Pol II by performing protein co-immunoprecipitation (Figure 6). For this, we used both a wild-type and a bud27Δ mutant strain that expressed a functional Myc-tagged version of Sth1. To immunoprecipitate RNA pol II, we used 8WG16 antibodies raised against the CTD repeats of Rpb1, the largest subunit of this RNA polymerase. We found a very significant decrease in the amount of Sth1-Myc that co-immunoprecipitated with Rpb1. In addition, as the deletion of BUD27 leads to Rpb1 cytoplasmic accumulation (3), we analysed the levels of two phosphorylated forms of RNA pol II (CTD Ser5P and CTD Ser2P), accounting for elongating RNA pol II, in the immunoprecipitated preparations. Notably, these levels were not lower in bud27Δ than in the wild-type strain, and the ratios Sth1-Myc:CTD Ser2P and Sth1-Myc:CTD Ser5P in the bud27Δ mutant were as low as the Sth1-Myc:Rpb1 values (Figure 6A). These results were corroborated by performing similar analyses in chromatin fractions of the same strains (Supplementary Figure S4A). In addition, a sharp increase, at least in the Ser5P:Rpb1 ratio was found in bud27Δ with respect to a wild-type strain (Figure 6B), supporting a role by Bud27 during early elongation. Again, these data were corroborated in chromatin fractions (Supplementary Figure S4B). We conclude that Bud27 is necessary to maintain a correct association of RSC to RNA pol II during transcription. Overall, our results show a functional contribution of Bud27 to RNA pol II-dependent transcription that may be mediated by its role in promoting the interaction between the RSC complex and Rpb5, in the context of the elongating RNA polymerase.

Bottom Line: In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation.We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II.Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén, Paraje de las Lagunillas, s/n, 23071, Jaén, Spain.

Show MeSH
Related in: MedlinePlus