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RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells.

Hsin JP, Li W, Hoque M, Tian B, Manley JL - Elife (2014)

Bottom Line: Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y).Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro.Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, United States.

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Complementation test and western blotting analysis in cells with Y1F mutations.(A) Complementation test was performed as described and results were shown. (B) Western blotting analysis of 20F+Y cells. Cells (20F+6Y) expressing an Rpb1 with the last 6 Y1F repeats replaced with normal YSPTSPS repeats were treated with tet for 24 hr. Rpb1 proteins were detected using Flag antibody. (C) Whole-cell lysates prepared from cells expressing the indicated Rpb1 derivative and treated with tet for 24 hr were analyzed by western blotting. Western blots were quantified using ImageJ, and % degradation from 3–4 independent replicates is presented. Error bars denote standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.004
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fig1s1: Complementation test and western blotting analysis in cells with Y1F mutations.(A) Complementation test was performed as described and results were shown. (B) Western blotting analysis of 20F+Y cells. Cells (20F+6Y) expressing an Rpb1 with the last 6 Y1F repeats replaced with normal YSPTSPS repeats were treated with tet for 24 hr. Rpb1 proteins were detected using Flag antibody. (C) Whole-cell lysates prepared from cells expressing the indicated Rpb1 derivative and treated with tet for 24 hr were analyzed by western blotting. Western blots were quantified using ImageJ, and % degradation from 3–4 independent replicates is presented. Error bars denote standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.004

Mentions: We previously utilized chicken DT40 cells to study properties of the Rpb1 CTD. We showed that an Rpb1 derivative containing a CTD with 26 YSPTSPS repeats (Rpb1-26r) plus the ten C-terminal non-consensus residues, important for stability (Chapman et al., 2005), confers cell viability, while a comparable derivative with all Thr4 residues changed to Val was inviable (Hsin et al., 2011). To investigate the functions of Tyr1, we constructed a plasmid encoding a Flag-tagged Rpb1 derivative, Rpb1-Y1F, identical to Rpb1-26r but with all Tyr1 residues replaced by Phe, and expressed this in Rpb1 conditional knock-out cells (DT40-Rpb1; Hsin et al., 2011). Tyr1 was suggested to be essential for viability in S. cerevisiae (West and Corden, 1995), but not in S. pombe (Schwer and Shuman, 2011). To determine whether Tyr1 is required for growth in vertebrate cells, DT40-Rpb1 cells were transfected with the Rpb1-Y1F vector, and tetracycline (tet) was added to turn off wild-type Rpb1 expression. Rpb1-Y1F was unable to complement Rpb1, whereas Rpb1-26r fully restored viability (Figure 1—figure supplement 1A).


RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells.

Hsin JP, Li W, Hoque M, Tian B, Manley JL - Elife (2014)

Complementation test and western blotting analysis in cells with Y1F mutations.(A) Complementation test was performed as described and results were shown. (B) Western blotting analysis of 20F+Y cells. Cells (20F+6Y) expressing an Rpb1 with the last 6 Y1F repeats replaced with normal YSPTSPS repeats were treated with tet for 24 hr. Rpb1 proteins were detected using Flag antibody. (C) Whole-cell lysates prepared from cells expressing the indicated Rpb1 derivative and treated with tet for 24 hr were analyzed by western blotting. Western blots were quantified using ImageJ, and % degradation from 3–4 independent replicates is presented. Error bars denote standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.004
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Related In: Results  -  Collection

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fig1s1: Complementation test and western blotting analysis in cells with Y1F mutations.(A) Complementation test was performed as described and results were shown. (B) Western blotting analysis of 20F+Y cells. Cells (20F+6Y) expressing an Rpb1 with the last 6 Y1F repeats replaced with normal YSPTSPS repeats were treated with tet for 24 hr. Rpb1 proteins were detected using Flag antibody. (C) Whole-cell lysates prepared from cells expressing the indicated Rpb1 derivative and treated with tet for 24 hr were analyzed by western blotting. Western blots were quantified using ImageJ, and % degradation from 3–4 independent replicates is presented. Error bars denote standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.004
Mentions: We previously utilized chicken DT40 cells to study properties of the Rpb1 CTD. We showed that an Rpb1 derivative containing a CTD with 26 YSPTSPS repeats (Rpb1-26r) plus the ten C-terminal non-consensus residues, important for stability (Chapman et al., 2005), confers cell viability, while a comparable derivative with all Thr4 residues changed to Val was inviable (Hsin et al., 2011). To investigate the functions of Tyr1, we constructed a plasmid encoding a Flag-tagged Rpb1 derivative, Rpb1-Y1F, identical to Rpb1-26r but with all Tyr1 residues replaced by Phe, and expressed this in Rpb1 conditional knock-out cells (DT40-Rpb1; Hsin et al., 2011). Tyr1 was suggested to be essential for viability in S. cerevisiae (West and Corden, 1995), but not in S. pombe (Schwer and Shuman, 2011). To determine whether Tyr1 is required for growth in vertebrate cells, DT40-Rpb1 cells were transfected with the Rpb1-Y1F vector, and tetracycline (tet) was added to turn off wild-type Rpb1 expression. Rpb1-Y1F was unable to complement Rpb1, whereas Rpb1-26r fully restored viability (Figure 1—figure supplement 1A).

Bottom Line: Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y).Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro.Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, United States.

Show MeSH