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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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Bud27 modulates transcription elongation. (A) Upper panel, transcription units used in this work. Lower panel, GLAM assay in wild-type and bud27Δ cells. (B) Growth of wild-type and bud27Δ strains in media containing drugs MPA and 6-AZA, at 30ºC in SD medium without uracil. (C) GLAM assay in wild-type cells under depletion of RPB5 by adding doxycycline at the indicated concentrations (Dox 0, Dox 1 and Dox 5 correspond to 0, 1 and 5 μg doxycycline/ml). (D) Chromatin fractions of a strain expressing a Bud27-TAP tagged construction from its own locus and its isogenic wild-type counterpart. Strains were grown in YPD medium at 30ºC. TAP (PAP), histone H3 and phosphoglycerate kinase (22C5D8) were analysed by western blot. (E) Rpb1 (8WG16), Rpb1-CTDSer5P (CTD4H8), Rpb1-CTDSer2P (ab5095), Bud27-TAP and Imd2-TAP were analysed by western in TAP purified complexes with either TAP-tagged Bud27 or Imd2 from cells growing in YPD medium at 30ºC. No tag: TAP purification with negative control extracts containing untagged Bud27.
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Figure 3: Bud27 modulates transcription elongation. (A) Upper panel, transcription units used in this work. Lower panel, GLAM assay in wild-type and bud27Δ cells. (B) Growth of wild-type and bud27Δ strains in media containing drugs MPA and 6-AZA, at 30ºC in SD medium without uracil. (C) GLAM assay in wild-type cells under depletion of RPB5 by adding doxycycline at the indicated concentrations (Dox 0, Dox 1 and Dox 5 correspond to 0, 1 and 5 μg doxycycline/ml). (D) Chromatin fractions of a strain expressing a Bud27-TAP tagged construction from its own locus and its isogenic wild-type counterpart. Strains were grown in YPD medium at 30ºC. TAP (PAP), histone H3 and phosphoglycerate kinase (22C5D8) were analysed by western blot. (E) Rpb1 (8WG16), Rpb1-CTDSer5P (CTD4H8), Rpb1-CTDSer2P (ab5095), Bud27-TAP and Imd2-TAP were analysed by western in TAP purified complexes with either TAP-tagged Bud27 or Imd2 from cells growing in YPD medium at 30ºC. No tag: TAP purification with negative control extracts containing untagged Bud27.

Mentions: Previously, we have proposed a role for Rpb5 in the transition from transcription initiation to elongation (25). If Bud27 influences RNA pol II-dependent transcription in an Rpb5-mediated manner, we should expect transcription elongation defects in bud27Δ cells. To address this question, we first investigated whether Bud27 is able to influence transcription elongation. For this, we used the length-dependent mRNA accumulation (GLAM) assay, commonly employed to detect defects in transcription elongation (22). As shown in Figure 3A, the GLAM ratio for bud27Δ mutant was significantly reduced compared to that observed for the wild-type strain. Consistently, we found that the growth of bud27Δ cells was impaired in the presence of 6-azauracil (6AU) and MPA, two Nucleotide Triphosphate (NTP)-depleting drugs used to detect S. cerevisiae strains defective for transcription elongation (27,28) (Figure 3B). These results strongly suggest a transcription elongation defect in the bud27 strain.


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)

Bud27 modulates transcription elongation. (A) Upper panel, transcription units used in this work. Lower panel, GLAM assay in wild-type and bud27Δ cells. (B) Growth of wild-type and bud27Δ strains in media containing drugs MPA and 6-AZA, at 30ºC in SD medium without uracil. (C) GLAM assay in wild-type cells under depletion of RPB5 by adding doxycycline at the indicated concentrations (Dox 0, Dox 1 and Dox 5 correspond to 0, 1 and 5 μg doxycycline/ml). (D) Chromatin fractions of a strain expressing a Bud27-TAP tagged construction from its own locus and its isogenic wild-type counterpart. Strains were grown in YPD medium at 30ºC. TAP (PAP), histone H3 and phosphoglycerate kinase (22C5D8) were analysed by western blot. (E) Rpb1 (8WG16), Rpb1-CTDSer5P (CTD4H8), Rpb1-CTDSer2P (ab5095), Bud27-TAP and Imd2-TAP were analysed by western in TAP purified complexes with either TAP-tagged Bud27 or Imd2 from cells growing in YPD medium at 30ºC. No tag: TAP purification with negative control extracts containing untagged Bud27.
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Figure 3: Bud27 modulates transcription elongation. (A) Upper panel, transcription units used in this work. Lower panel, GLAM assay in wild-type and bud27Δ cells. (B) Growth of wild-type and bud27Δ strains in media containing drugs MPA and 6-AZA, at 30ºC in SD medium without uracil. (C) GLAM assay in wild-type cells under depletion of RPB5 by adding doxycycline at the indicated concentrations (Dox 0, Dox 1 and Dox 5 correspond to 0, 1 and 5 μg doxycycline/ml). (D) Chromatin fractions of a strain expressing a Bud27-TAP tagged construction from its own locus and its isogenic wild-type counterpart. Strains were grown in YPD medium at 30ºC. TAP (PAP), histone H3 and phosphoglycerate kinase (22C5D8) were analysed by western blot. (E) Rpb1 (8WG16), Rpb1-CTDSer5P (CTD4H8), Rpb1-CTDSer2P (ab5095), Bud27-TAP and Imd2-TAP were analysed by western in TAP purified complexes with either TAP-tagged Bud27 or Imd2 from cells growing in YPD medium at 30ºC. No tag: TAP purification with negative control extracts containing untagged Bud27.
Mentions: Previously, we have proposed a role for Rpb5 in the transition from transcription initiation to elongation (25). If Bud27 influences RNA pol II-dependent transcription in an Rpb5-mediated manner, we should expect transcription elongation defects in bud27Δ cells. To address this question, we first investigated whether Bud27 is able to influence transcription elongation. For this, we used the length-dependent mRNA accumulation (GLAM) assay, commonly employed to detect defects in transcription elongation (22). As shown in Figure 3A, the GLAM ratio for bud27Δ mutant was significantly reduced compared to that observed for the wild-type strain. Consistently, we found that the growth of bud27Δ cells was impaired in the presence of 6-azauracil (6AU) and MPA, two Nucleotide Triphosphate (NTP)-depleting drugs used to detect S. cerevisiae strains defective for transcription elongation (27,28) (Figure 3B). These results strongly suggest a transcription elongation defect in the bud27 strain.

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