Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

Abstract

Gpn1 associates with Gpn3, and both are required for RNA polymerase II nuclear targeting. Global studies have identified by mass spectrometry that human Gpn3 is ubiquitinated on lysines 189 and 216. Our goals here were to determine the type, physiological importance, and regulation of Gpn3 ubiquitination. After inhibiting the proteasome with MG132, Gpn3-Flag was polyubiquitinated on K216, but not K189, in HEK293T cells. Gpn3-Flag exhibited nucleo-cytoplasmic shuttling, but polyubiquitination and proteasomal degradation of Gpn3-Flag occurred only in the cell nucleus. Polyubiquitination-deficient Gpn3-Flag K216R displayed a longer half-life than Gpn3-Flag in two cell lines. Interestingly, Gpn1-EYFP inhibited Gpn3-Flag polyubiquitination in a dose-dependent manner. In conclusion, Gpn1-inhibitable, nuclear polyubiquitination on lysine 216 regulates the half-life of Gpn3 by tagging it for proteasomal degradation.

Npa3 interacts with Gpn3 and assembly factor Rba50 for RNA polymerase II biogenesis

“…Gpn1, Gpn2, and Gpn3 are all essential proteins for cell growth, and deletion of each one results in cell death, suggesting that their functions are not redundant. Interestingly, Gpn1 and Gpn3 in mammalian cells are closely associated for RNAPII biogenesis regarding to protein level stability, mutual dependency, and genetic interaction 21‐24 . In the previous study, we identified yeast NPA3 and GPN3 as two multicopy genetic suppressors of gpn2 ts mutants 14 .…”

“…Interestingly, Gpn1 and Gpn3 in mammalian cells are closely associated for RNAPII biogenesis regarding to protein level stability, mutual dependency, and genetic interaction. [21][22][23][24] In the previous study, we identified yeast NPA3 and GPN3 as two multicopy genetic suppressors of gpn2 ts mutants. 14 In the same screen, we obtained RBA50 as a strong suppressor of gpn2 ts as well, and demonstrated that Rba50 is an essential co-factor of Gpn2 in the process of Rpb3-dependent assembly.…”