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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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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 sense strand RNAs (p<0.05, Fisher's exact test) were highlighted. The five genes validated in Figure 4C were marked in the plot. (B) Expression of antisense poly(A)+ RNA near the transcription start site (TSS) in 26r and 25F+Y cells. Reads per million (RPM) per base for poly(A) sites were shown (y-axis). All genes with upstream antisense (ua) RNAs detected in either 26r or 25F+Y cells were used for plotting. The curves were smoothened by the ‘lowess’ function.DOI:http://dx.doi.org/10.7554/eLife.02112.012
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fig4s3: 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 sense strand RNAs (p<0.05, Fisher's exact test) were highlighted. The five genes validated in Figure 4C were marked in the plot. (B) Expression of antisense poly(A)+ RNA near the transcription start site (TSS) in 26r and 25F+Y cells. Reads per million (RPM) per base for poly(A) sites were shown (y-axis). All genes with upstream antisense (ua) RNAs detected in either 26r or 25F+Y cells were used for plotting. The curves were smoothened by the ‘lowess’ function.DOI:http://dx.doi.org/10.7554/eLife.02112.012

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)

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 sense strand RNAs (p<0.05, Fisher's exact test) were highlighted. The five genes validated in Figure 4C were marked in the plot. (B) Expression of antisense poly(A)+ RNA near the transcription start site (TSS) in 26r and 25F+Y cells. Reads per million (RPM) per base for poly(A) sites were shown (y-axis). All genes with upstream antisense (ua) RNAs detected in either 26r or 25F+Y cells were used for plotting. The curves were smoothened by the ‘lowess’ function.DOI:http://dx.doi.org/10.7554/eLife.02112.012
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fig4s3: 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 sense strand RNAs (p<0.05, Fisher's exact test) were highlighted. The five genes validated in Figure 4C were marked in the plot. (B) Expression of antisense poly(A)+ RNA near the transcription start site (TSS) in 26r and 25F+Y cells. Reads per million (RPM) per base for poly(A) sites were shown (y-axis). All genes with upstream antisense (ua) RNAs detected in either 26r or 25F+Y cells were used for plotting. The curves were smoothened by the ‘lowess’ function.DOI:http://dx.doi.org/10.7554/eLife.02112.012
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