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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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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 YSPTAPS repeats. The number of reads mapped to polyA sites for each cell type are shown.DOI:http://dx.doi.org/10.7554/eLife.02112.010
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fig4s1: 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 YSPTAPS repeats. The number of reads mapped to polyA sites for each cell type are shown.DOI:http://dx.doi.org/10.7554/eLife.02112.010

Mentions: We next wished to determine the genome-wide impact of the 25F+Y mutation on transcript levels. Using 3′READS (Hoque et al., 2013), a deep sequencing method to quantitate poly(A)+ RNAs, we analyzed 25F+Y and 26r cells, as well as S2A, S5A and T4V cells (all of which, like 25F+Y, are inviable; Hsin et al., 2011; Hsin et al., 2014) for comparison. Cells were treated with tet for 24 hr, and a total of ∼5 million reads mapping to 3′ regions of genes were generated for each cell type (Figure 4—figure supplement 1). Reads were classified into sense RNAs and upstream antisense (ua) RNAs (Figure 4A). uaRNAs were defined as transcripts that did not overlap any known protein-coding genes and used a poly(A) site within 2 kb from the TSS (Figure 4—figure supplement 2). Unexpectedly, the number of genes with upregulated uaRNAs was significantly greater than the number of genes with downregulated uaRNAs, by ∼16-fold (p=10−21.5), in 25F+Y cells (Figure 4B). S2A and S5A cells showed similar trends but to much lesser extents, fourfold (p=10−6.7) and 5.6-fold (p=10−9.0), respectively, while T4V cells in fact showed a trend in the opposite direction (Figure 4B). Using RT-qPCR, we validated several of the uaRNAs (Figure 4C). uaRNAs associated with the ARGLU1, METTL14, SH3BP5 and WEE1 genes were upregulated about twofold in two independent 25F+Y cell lines, consistent with results from RNA-seq (Figure 4—figure supplement 3A). Levels of RPLP1- and CCNB2-associated uaRNAs were indistinguishable in 26r and 25F+Y cells by both methods.10.7554/eLife.02112.009Figure 4.Tyr1 functions in expression of upstream antisense transcripts.


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

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

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 YSPTAPS repeats. The number of reads mapped to polyA sites for each cell type are shown.DOI:http://dx.doi.org/10.7554/eLife.02112.010
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042873&req=5

fig4s1: 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 YSPTAPS repeats. The number of reads mapped to polyA sites for each cell type are shown.DOI:http://dx.doi.org/10.7554/eLife.02112.010
Mentions: We next wished to determine the genome-wide impact of the 25F+Y mutation on transcript levels. Using 3′READS (Hoque et al., 2013), a deep sequencing method to quantitate poly(A)+ RNAs, we analyzed 25F+Y and 26r cells, as well as S2A, S5A and T4V cells (all of which, like 25F+Y, are inviable; Hsin et al., 2011; Hsin et al., 2014) for comparison. Cells were treated with tet for 24 hr, and a total of ∼5 million reads mapping to 3′ regions of genes were generated for each cell type (Figure 4—figure supplement 1). Reads were classified into sense RNAs and upstream antisense (ua) RNAs (Figure 4A). uaRNAs were defined as transcripts that did not overlap any known protein-coding genes and used a poly(A) site within 2 kb from the TSS (Figure 4—figure supplement 2). Unexpectedly, the number of genes with upregulated uaRNAs was significantly greater than the number of genes with downregulated uaRNAs, by ∼16-fold (p=10−21.5), in 25F+Y cells (Figure 4B). S2A and S5A cells showed similar trends but to much lesser extents, fourfold (p=10−6.7) and 5.6-fold (p=10−9.0), respectively, while T4V cells in fact showed a trend in the opposite direction (Figure 4B). Using RT-qPCR, we validated several of the uaRNAs (Figure 4C). uaRNAs associated with the ARGLU1, METTL14, SH3BP5 and WEE1 genes were upregulated about twofold in two independent 25F+Y cell lines, consistent with results from RNA-seq (Figure 4—figure supplement 3A). Levels of RPLP1- and CCNB2-associated uaRNAs were indistinguishable in 26r and 25F+Y cells by both methods.10.7554/eLife.02112.009Figure 4.Tyr1 functions in expression of upstream antisense transcripts.

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