Human U3 protein14a is a novel type ubiquitin ligase that binds RB and promotes RB degradation depending on a leucine-rich region.

The nucleolar protein hUTP14a promotes p53 degradation and possesses an oncogene potential. Here, we report that hUTP14a promotes degradation of tumor suppressor retinoblastoma (RB) protein. Sequences alignment showed that hUTP14a contains the RB-binding PENF motif in its C-terminus. We showed that hUTP14a interacted with RB in vivo and in vitro. Further, hUTP14a promoted polyubiquitination and proteasome-dependent turnover of RB. Importantly, purified Flag-hUTP14a facilitated RB ubiquitination in vitro, demonstrating that hUTP14a is an ubiquitin E3 ligase for RB. A BLAST alignment with hUTP14a does not reveal a RING or HECT domain. To define the conserved domain for E3 ligase activity in hUTP14a, the minimum domain for promoting RB degradation was mapped to residues 61-120 of hUTP14a, in which a leucine-rich region (LRR) LxLxxLL was suggested to be conserved. Flag-hUTP14a (ΔLRR), Flag-hUTP14a-MT1(LxLxxLL to LxLxxAA) and Flag-hUTP14a-MT2(LxLxxLL to AxAxxAA) lost the capability of ubiquitinating RB in vitro, demonstrating that LRR is required for the E3 ligase activity of hUTP14a. Consequently, expression of hUTP14a caused upregulation of E2F1 downstream genes, thus promoting cancer cell proliferation. Taken together, we demonstrate that hUTP14a promotes RB degradation through its E3 ligase activity and suggest that the LRR could be a potential conserved E3 ligase domain.