RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells
- Columbia University, United States
- Rutgers University New Jersey Medical School, United States
Figures
Figure 1 with 1 supplement
Growth properties of Rpb1 cell lines.
(A) Cells were cultured in medium containing 1 µg/ml tetracycline (tet). Control cells, 26r, were split on day 2. Average cell counts from two independent experiments were plotted. (B) Cells were …
Figure 1—figure supplement 1
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 …
Figure 2 with 1 supplement
Rpb1 Tyr1 phosphorylation is found in all cell fractions.
(A) Flag-tagged Rpb1 proteins were immunoprecipitated from cells treated with tet for 24 hr, and analyzed using western blotting. Phosphorylation on Tyr1 (Tyr1-P) was detected by the 3D12 antibody. …
Figure 2—figure supplement 1
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 …
Figure 3 with 1 supplement
Tyr1 phosphorylation functions in CTD stability.
(A) In vitro 20S proteasome assay. 200 nM purified GST-CTD was incubated with 5 nM bovine 20S proteasome, and the reaction was carried with or without 0.01% SDS. MG132 was used to inhibit the …
Figure 3—figure supplement 1
Western blotting analysis and quantification.
(A) GST-CTD proteins were phosphorylated by Abl tyrosine kinase for indicated time in vitro. Reactions were analyzed by western blotting with indicated antibodies. (B) Y1F cells grown in the absence …
Figure 4 with 6 supplements
Tyr1 functions in expression of upstream antisense transcripts.
(A) Schematic of sense RNA and upstream antisense (ua) RNAs analyzed. (B) Regulation of uaRNA expression. Cells treated with tet for 24 hr were processed for 3′READS RNA-seq analysis. The number of …
Figure 4—figure supplement 1
RNA-Seq analysis of Rpb1 cell lines.
RNA from cells treated with tet for 24 hr were processed for deep sequencing as described. S2A cells express an Rpb1 derivative with 26 YAPTSPS repeats, whereas S5A cells express an Rpb1 with 28 …
Figure 4—figure supplement 2
uaRNA polyA site analysis.
(A) The distribution of uaRNA polyA sites. The number of polyA sites in generated reads was counted and plotted against their distance from TSS. (B) The nucleotide profiles of polyA sites in (A).
Figure 4—figure supplement 3
uaRNA expression analysis in 25F+Y and 26r cells.
(A) Expression difference of uaRNAs vs sense strand RNAs for 25F+Y and 26r cells. Each dot is a gene with uaRNA expression detected. Genes with significant difference in expression of uaRNAs vs …
Figure 4—figure supplement 4
Exosome subunit levels.
Cells were treated with tet for 24 hr. The levels of exosome subunits Exosc 3 (Rrp40), Exosc 9 (Rrp45), Exosc 10 (Rrp6), and Dis3 were determined by western blotting.
Figure 4—figure supplement 5
Expression changes for different types of transcripts in 25F+Y cells compared to 26r cells.
Values are shown in cumulative distribution function (CDF) curves. Transcript type is indicated in the graph. Expression change was based on log2 ratio of the Read Per Million total PASS reads (RPM) …
Figure 4—figure supplement 6
Tyr1-P ChIP analysis.
Data from Figure 4D were reanalyzed by normalizing the Tyr1-P signals to Rpb1 levels. N = 3. Error bars display standard deviation.
Additional files
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Supplementary file 1
List of primer sequences.
- https://doi.org/10.7554/eLife.02112.016
