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fig4s4: 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.DOI:http://dx.doi.org/10.7554/eLife.02112.013
Mentions: We next investigated the mechanism underling the enhanced accumulation of uaRNAs in 25F+Y cells. uaRNAs are low-abundant, usually rapidly degraded by the nuclear exosome (Preker et al., 2008; Seila et al., 2009; Wei et al., 2011; Ntini et al., 2013). However, protein levels of four exosome subunits were comparable in 26r and 25F+Y cells (Figure 4—figure supplement 4), suggesting that the increase in uaRNAs was unlikely due to decreased exosome levels. Also, poly(A) sites of uaRNAs were unchanged (Figure 4—figure supplement 3B), indicating that enhanced accumulation did not reflect altered poly(A) site utilization. Another possibility was that transcription of these transcripts was increased. However, ChIP assays indicated that Rpb1 levels were in fact reduced upstream of the ARGLU1, METTL14, SH3BP5 and WEE1 genes in 25F+Y cells (Figure 4D; see also Figure 4—figure supplement 5). Finally, ChIP analyses showed more Tyr1-P on these upstream genes than on the corresponding downstream sense genes (Figure 4—figure supplement 6). Our results point to a role for Tyr1-P in regulating accumulation of uaRNAs by contributing to their rapid turnover.
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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