The TFIIH Subunit Tfb3 Regulates Cullin Neddylation

Abstract

Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins.

Protein neddylation: beyond cullin–RING ligases

“…It is conceivable that different DCNLs regulate different cullins in vivo . For example the only known DCNL orthologue in budding yeast, Dcn1, is important for the neddylation of Cul1 and Cul3, but not the CUL4 orthologue Rtt101 70 . The human DCNL paralogues differ in subcellular localization.…”

“…Tfb3 comprises a RING domain, which regulates the neddylation of CUL3 and CUL4 but not CUL1 orthologues 70 . Similarly to RBX1, Tfb3 interacts with Ubc12 and lacks the lynchpin arginine (Figure 2D), which indicates that it may directly activate Ubc12.…”

“…For example, the yeast CUL4-orthologue Rtt101 can be neddylated or ubiquitinated, both leading to its catalytic activation 70 . Moreover, it is possible that poly-neddylation and poly-ubiquitylation at DNA damage sites or in response to other stress conditions are functionally redundant, as NEDD8 and ubiquitin can be recognized by the same interaction motifs.…”

Ubiquitin and Proteasomes in Transcription

“…TAF I , for example, is a core component of the TFIID complex and is unusual in that it has combined E1 and E2 activities (95), allowing it to single-handedly activate Ub and conjugate it to a substrate. The basal factor TFIIH has two potential ways it can influence ubiquitylation, acting as a direct Ub ligase (96) and by modulating the activity of other ligases that are regulated by the Ub-like modification Nedd8 (97). And as mentioned above, the SAGA chromatin-remodeling complex has a built in DUb (88) that deubiquitylates H2B, and potentially other substrates.…”

Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5

“…Vice versa, mammalian CSN is able to deneddylate yeast cullins 4,5 . However, the knockdown of the yeast CSN subunits neither affect viability nor the G1-S transition promoted by the SCF, and it has a modest effect on the turnover of SCF substrates [6][7][8] .…”