EL VÍNCULO SOCIO-AFECTIVO

TRANSCRIPTIONALLY STALLED RNAPII.

After polyubiquitylation of the substrate targeted for degradation, the 26S proteasome is responsible for its proteolysis. However, in the case of transcriptional stalling it is possible that through the function of general transcription elongation factors the stalled complex escapes arrest and is then able to restart transcription. Since RNAPII is an important enzyme a rescue mechanism must exist to prevent unnecessary Rpb1 degradation in complexes that are not stalled any more. So, ‘‘proofreading’’ of Rpb1 ubiquitylation by deubiquitylases (DUBs) is crucial for cell viability. This “proofreading” could serve for delaying RNAPII degradation until transcription factors can help the stalled complex to resume transcription by maintaining short-length polyubiquitin chain. Additionally, deubiquitylation by the DUBs could serve for preventing degradation of a rescued from staling RNAPII complex by cleaving off the polyubiquitin chain from Rpb1 to produce completely deubiquitylated RNAPII. S.cerevisiae cells contain 17–20 DUBs, 16 of which belong to the specific ubiquitin protease (UBPs) family. The UBPs antagonize the ubiquitylation of proteins, playing a role analogous to that of the phosphatases in a kinase/phosphatase regulatory pathway (see Introduction 1.7.3).

In the DNA damage-dependent pathway there are two deubiquitylases that have been described to be involved: Ubp2 deubiquitylates excess K63-linked polyubiquitin chains

mediated K48-linked polyubiquitin chains from Rpb1 (Kvint, Uhler et al. 2008; Harreman, Taschner et al. 2009). These two proteins along with 3 more enzymes (Ubp6, Ubp10 and Ubp12) were selected based on their sensitivity to 6AU (Kvint, Uhler et al. 2008) to be tested for their possible function in deubiquitylating Rpb1 in response to transcriptional stalling. To this end, yeast strains were created that lacked each of these enzymes either alone or in combination with the deletion of DST1. All the double mutants were viable and no additive growth defects were observed (Figure 39A).

Figure 39 | The UBPs Ubp2, 3, 6, 10,12 do not affect the growth of ∆_dst1 cells and the total cellular levels of Rpb1.

A, Growth analysis of the single and double deletion mutants of DST1 and the different UBPs. No synthetic lethality phenotype was observed. B, Total cellular Rpb1 levels were quantified in the indicated strains. Experiment as in Figure 12. No significant change was observed in the double mutants. Columns and error bars represent the mean ± standard deviation from 3 independent experiments

The UBPs are known to affect the stability of ubiquitylated proteins (Chung and Baek 1999; Wilkinson 2000). To investigate the effect of the deletion of the UBPs in RNAPII levels, the protein levels of Rpb1 were quantified in the single and double deletion strains. As shown in Figure 39B, there was no increased degradation of Rpb1 in any of the UBPs tested. This indicates that deletion of the deubiquitylases does not reduce the half-life of polyubiquitylated Rpb1.

To investigate which of the candidate UBPs is involved in deubiquitylating RNAPII in the transcriptional stalling-dependent pathway the level of polyubiquitylated Rpb1 was assessed in the single and the double deletion mutants. Consistent with a function of Rsp5 in ubiquitylating Rpb1 of transcriptionally stalled RNAPII complexes, Ubp2 is required for deubiquitylation of Rpb1 (Figure 40, compare lanes 2 and 6). However, Ubp3 is not required for deubiquitylating Rpb1 since additional deletion of the protein in the ∆dst1

background did not result in an increase in polyubiquitylation of Rpb1 (Figure 40, compare lanes 2 and 8). Importantly, whereas Ubp10 and Ubp12 are not required (Figure 40, compare lanes 2 with 10 and 12), Ubp6 was identified to be responsible for deubiquitylating Rpb1 upon transcriptional stalling (Fig. 40, compare lanes 1 to 4).

Figure 40 | Ubp2 and Ubp6 are required for deubiquitylation of transcriptionally stalled Rpb1. RNAPII was purified from the indicated strains. Experiment as in Figure 20. Polyubiquitylation of Rpb1 is increased, i.e. deubiquitylation of Rpb1 is decreased in Δdst1 cells that also carry a deletion in the Ubp6 and Ubp2 deubiquitylases. However, there is no effect observed upon deletion of Ubp3, Ubp10 and Ubp12.

Ubp6, a cysteine protease, is an abundant proteasome-associated protein, which associates with the base of the proteasome. Binding of Ubp6 to the proteasome activates Ubp6’s catalytic activity (Leggett, Hanna et al. 2002), indicating an intimate functional relationship between Ubp6 and the proteasome. Recently it was reported that Ubp6 delays the breakdown of proteins by the proteasome. During this degradation delay, substrate deubiquitylation proceeds on proteasomes, but with a different mode than the one observed in the absence of Ubp6 (Hanna, Hathaway et al. 2006). Overall, it was suggested that Ubp6 has a catalytic as well as a non-catalytic function. Specifically, it deubiquitylates ubiquitylated proteins before their degradation thereby recycling ubiquitin and it delays degradation of polyubiquitylated proteins by the proteasome in a mainly non-catalytic manner (Hanna, Hathaway et al. 2006). To determine whether the catalytic activity of Ubp6 is indeed needed for deubiquitylation of Rpb1 (Figure 40) we assessed Rpb1 ubiquitylation in the catalytically inactive ubp6-C118A mutant that still inhibits the proteasome (Hanna, Hathaway et al. 2006). Rpb1 ubiquitylation levels are increased in

ubp6-C118A Δdst1 cells to a similar extent as in Δubp6 Δdst1 cells showing that the deubiquitylase activity of Ubp6 is necessary for Rpb1 deubiquitylation (Figure 41).

Figure 41 | The deubiquitylating activity of Ubp6 is required for Rpb1 deubiquitylation.

RNAPII was purified from the indicated strains. Experiment as in Figure 20. Polyubiquitylation of Rpb1 is increased, i.e. deubiquitylation of Rpb1 is decreased in Δdst1 cells that also carry either a deletion or a catalytic- inactive mutation of Ubp6. Columns and error bars represent the mean ± standard deviation from 3 independent experiments.

Taken together, two proteins, Ubp2 and Ubp6, are responsible for deubiquitylating Rpb1 of transcriptionally stalled RNAPII complexes.

2.8

SPECIFIC DEGRADATION UPON TRANSCRIPTIONAL IMPAIRMENT MIGHT