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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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Western blotting analysis.(A) Cells were treated with tet for 24 hr, and then subjective to subcellular fractionation. Rpb1 localization was determined by western blotting. Nuclear protein U2AF65, and chromatin-bound histone H3 served as controls for fractionation. Asterisk (*) indicates the degraded Rpb1 fragment. (B) Subcellular fractionation assay was performed in HEK293 cells. The localization of Rpb1 phosphorylated on Tyr1, Ser 2, 5, 7, and Thr4 was determined using antibodies as described in ‘Materials and methods’. (C) Rpb1 from DT40 cell lysates were immunoprecipitated using antibodies recognizing phosphoserine 5 (3E8) or phosphoserine 2 (3E10). The association of tyrosine phosphorylation with phosphoserine 5 or phosphoserine 2 was determined by western blotting using the 3D12 antibody.DOI:http://dx.doi.org/10.7554/eLife.02112.006
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fig2s1: Western blotting analysis.(A) Cells were treated with tet for 24 hr, and then subjective to subcellular fractionation. Rpb1 localization was determined by western blotting. Nuclear protein U2AF65, and chromatin-bound histone H3 served as controls for fractionation. Asterisk (*) indicates the degraded Rpb1 fragment. (B) Subcellular fractionation assay was performed in HEK293 cells. The localization of Rpb1 phosphorylated on Tyr1, Ser 2, 5, 7, and Thr4 was determined using antibodies as described in ‘Materials and methods’. (C) Rpb1 from DT40 cell lysates were immunoprecipitated using antibodies recognizing phosphoserine 5 (3E8) or phosphoserine 2 (3E10). The association of tyrosine phosphorylation with phosphoserine 5 or phosphoserine 2 was determined by western blotting using the 3D12 antibody.DOI:http://dx.doi.org/10.7554/eLife.02112.006

Mentions: We next set out to determine how Tyr1 residues stabilize Rpb1. A first question was whether Rpb1 is indeed Tyr1-phosphorylated in DT40 cells. To address this, we utilized an anti-phospho-Tyr1 Ab (Mayer et al., 2012) to examine Tyr1 phosphorylation (Tyr1-P) of Rpb1-25F+Y and Rpb1-26r by WB; both proteins were indeed Tyr1-phosphorylated (Figure 2A). We next investigated where in cells the Rpb1-b isoform accumulates. We analyzed cytoplasmic, nuclear and chromatin-bound fractions from 26r and Y1F cells by WB with an N-terminal Rpb1 Ab (N20). Rpb1-b (indicated by *) was detected in all three fractions from Y1F cells, but barely or not at all in the 26r fractions (Figure 2B). The relative (and absolute) Rpb1-b levels were lowest in the cytoplasm, while Rpb1-b was essentially the only form on Y1F chromatin. As anticipated, Rpb1-b was not detected in 25F+Y cell fractions (Figure 2—figure supplement 1A). We next determined whether Tyr1-P could also be detected on Rpb1 in all three fractions, in this case using extracts from wild-type DT40 (Figure 2C) and human HEK293 (Figure 2—figure supplement 1B) cells. Robust Tyr1-P was indeed detected in all three fractions in both cell types. Notably, in both cytoplasm and nucleoplasm, Tyr1-P was observed only on hypophosphorylated Rpb1 (the lower band), while it was found primarily on the hyperphosphorylated isoform on chromatin. This suggests both that CTD phosphorylation is limited to Tyr1 in the cytoplasm and nucleoplasm and that Tyr1-P is present on hyperphosphorylated RNAP II found on active genes. We also examined phosphorylation on Ser 2, 5 and 7 and Thr4 (Figure 2C, Figure 2—figure supplement 1B). All these modifications were nearly undetectable in cytoplasmic and nuclear fractions, present almost exclusively on chromatin-associated, hyperphosphorylated Rpb1. Together, our data show that Tyr1, and only Tyr1, is phosphorylated before RNAP II engages in transcription, and support the idea that Tyr1-P functions in stabilizing the CTD when RNAP II is not transcribing, and perhaps also plays a role during transcription. Consistent with this, Tyr1-P was detected on Rpb1 immunoprecipitated by Abs recognizing Ser5-P and Ser2-P (Figure 2—figure supplement 1C).10.7554/eLife.02112.005Figure 2.Rpb1 Tyr1 phosphorylation is found in all cell fractions.


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

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

Western blotting analysis.(A) Cells were treated with tet for 24 hr, and then subjective to subcellular fractionation. Rpb1 localization was determined by western blotting. Nuclear protein U2AF65, and chromatin-bound histone H3 served as controls for fractionation. Asterisk (*) indicates the degraded Rpb1 fragment. (B) Subcellular fractionation assay was performed in HEK293 cells. The localization of Rpb1 phosphorylated on Tyr1, Ser 2, 5, 7, and Thr4 was determined using antibodies as described in ‘Materials and methods’. (C) Rpb1 from DT40 cell lysates were immunoprecipitated using antibodies recognizing phosphoserine 5 (3E8) or phosphoserine 2 (3E10). The association of tyrosine phosphorylation with phosphoserine 5 or phosphoserine 2 was determined by western blotting using the 3D12 antibody.DOI:http://dx.doi.org/10.7554/eLife.02112.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2s1: Western blotting analysis.(A) Cells were treated with tet for 24 hr, and then subjective to subcellular fractionation. Rpb1 localization was determined by western blotting. Nuclear protein U2AF65, and chromatin-bound histone H3 served as controls for fractionation. Asterisk (*) indicates the degraded Rpb1 fragment. (B) Subcellular fractionation assay was performed in HEK293 cells. The localization of Rpb1 phosphorylated on Tyr1, Ser 2, 5, 7, and Thr4 was determined using antibodies as described in ‘Materials and methods’. (C) Rpb1 from DT40 cell lysates were immunoprecipitated using antibodies recognizing phosphoserine 5 (3E8) or phosphoserine 2 (3E10). The association of tyrosine phosphorylation with phosphoserine 5 or phosphoserine 2 was determined by western blotting using the 3D12 antibody.DOI:http://dx.doi.org/10.7554/eLife.02112.006
Mentions: We next set out to determine how Tyr1 residues stabilize Rpb1. A first question was whether Rpb1 is indeed Tyr1-phosphorylated in DT40 cells. To address this, we utilized an anti-phospho-Tyr1 Ab (Mayer et al., 2012) to examine Tyr1 phosphorylation (Tyr1-P) of Rpb1-25F+Y and Rpb1-26r by WB; both proteins were indeed Tyr1-phosphorylated (Figure 2A). We next investigated where in cells the Rpb1-b isoform accumulates. We analyzed cytoplasmic, nuclear and chromatin-bound fractions from 26r and Y1F cells by WB with an N-terminal Rpb1 Ab (N20). Rpb1-b (indicated by *) was detected in all three fractions from Y1F cells, but barely or not at all in the 26r fractions (Figure 2B). The relative (and absolute) Rpb1-b levels were lowest in the cytoplasm, while Rpb1-b was essentially the only form on Y1F chromatin. As anticipated, Rpb1-b was not detected in 25F+Y cell fractions (Figure 2—figure supplement 1A). We next determined whether Tyr1-P could also be detected on Rpb1 in all three fractions, in this case using extracts from wild-type DT40 (Figure 2C) and human HEK293 (Figure 2—figure supplement 1B) cells. Robust Tyr1-P was indeed detected in all three fractions in both cell types. Notably, in both cytoplasm and nucleoplasm, Tyr1-P was observed only on hypophosphorylated Rpb1 (the lower band), while it was found primarily on the hyperphosphorylated isoform on chromatin. This suggests both that CTD phosphorylation is limited to Tyr1 in the cytoplasm and nucleoplasm and that Tyr1-P is present on hyperphosphorylated RNAP II found on active genes. We also examined phosphorylation on Ser 2, 5 and 7 and Thr4 (Figure 2C, Figure 2—figure supplement 1B). All these modifications were nearly undetectable in cytoplasmic and nuclear fractions, present almost exclusively on chromatin-associated, hyperphosphorylated Rpb1. Together, our data show that Tyr1, and only Tyr1, is phosphorylated before RNAP II engages in transcription, and support the idea that Tyr1-P functions in stabilizing the CTD when RNAP II is not transcribing, and perhaps also plays a role during transcription. Consistent with this, Tyr1-P was detected on Rpb1 immunoprecipitated by Abs recognizing Ser5-P and Ser2-P (Figure 2—figure supplement 1C).10.7554/eLife.02112.005Figure 2.Rpb1 Tyr1 phosphorylation is found in all cell fractions.

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