Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

Abstract

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.Ubiquitin-dependent proteolysis involves the covalent ligation of ubiquitin to substrate proteins, which are then recognized and degraded by the 26S proteasome. While many of the components involved in catalyzing protein ubiquitination have been identified and characterized biochemically, we are only beginning to understand how the system specifically recognizes appropriate substrates. At least three classes of activities, known as E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-protein ligase) enzymes, cooperate in catalyzing protein ubiquitination (34). The enzymatic mechanisms and functions of the E1 and E2 proteins have been well characterized. In contrast, the E3 enzymes are a diverse and less-well-characterized group of activities, and many lines of evidence indicate that E3 activities play a major role in determining the substrate specificity of the ubiquitination pathway (14,28,34).The hect (homologous to E6-AP carboxyl terminus) domain defines a family of E3 proteins that were discovered through the characterization of human E6-AP (17). The interaction of E6-AP with the E6 protein of the cervical cancer-associated human papillomavirus types causes E6-AP to associate with and ubiquitinate p53, suggesting that E6 functions in promoting cellular immortalization by, at least in part, stimulating the destruction of this important tumor suppressor protein (16). The hect E3 molecular masses range from 92 to over 500 kDa, with the hect domain comprising the approximately 350 carboxyl-terminal amino acids (17, 34). Exactly five hect E3s are encoded by the Saccharomyces cerevisiae genome, and over 30 have been identified so far in mammalian species. An obligatory intermediate in the ubiquitination reactions catalyzed by hect E3s is a ubiquitin-thioester formed between the thiol group of an absolutely conserved cysteine within the hect domain and the terminal carboxyl group of ubiquitin (33). E3 becomes "charged" with ubiquitin vi...

The HECT E3 ubiquitin ligase Rsp5 is important for ubiquitin homeostasis in yeast

“…Ubiquitination by Rsp5 appears to play a role in DNA damage-induced degradation of Rbp1 [22]. Upon UV-irradiation, or nucleotide starvation, RNA polymerase complexes may become stalled on DNA.…”

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

“…Studies in mammalian systems [89,62] provided evidence that RNAPIIo is a target for ubiquitylation in UV-irradiated human cells and that ubiquitylation requires CSA npg and CSB (in contrast to the yeast CSB counterpart Rad26 that inhibits degradation of RNAPII [90]). However, recent results by Svejstrup and co-workers [91] suggest that Nedd4 is the ubiquitin ligase for damage-induced ubiquitylation of RNAPII, and that CS proteins are involved only indirectly.…”

von Hippel–Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination

“…In particular, ubiquitination of the hyperphosphorylated Rpb1 is induced by UV radiation and DNA damage (24)(25)(26), suggesting that Rpb1 ubiquitination may play a role in the transcription-coupled repair (27). In yeast, ubiquitination is mediated by a HECT-class Rsp5 ubiquitin ligase (28); however, the nature of the E3 ligase in mammalian cells is unknown. We hypothesized that the hyperphosphorylated Rpb1 may be a substrate for pVHL-associated E3 ubiquitin-ligase activity.…”