FRET‐based analysis and molecular modeling of the human GPN‐loop GTPases 1 and 3 heterodimer unveils a dominant‐negative protein complex

Abstract

GPN‐loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1–Gpn3 interaction in live HEK293‐AD cells. Transiently expressed EYFP‐Gpn1 and Gpn3‐CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293‐AD cells. Molecular modeling of the human Gpn1–Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α‐helix 7, insertion 2, and the GPN‐loop from each protein. In site‐directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP‐Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3‐CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293‐AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1–Gpn3 interaction is required to display the dominant‐negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1–Gpn3 strong interaction is critical for their cellular function.

Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small-cell lung cancer

“…BioPlex 2.0 (47) predicted an interaction between RPA1 and GPN3 ( Figure S8A). This was interesting because GPN3, through a physical association with the related protein GPN1, associates with the Pol II complex and in some cells promotes assembly of functional Pol II in the nucleus (48)(49)(50)(51). GPN1 and GPN3…”

Biogenesis of RNA Polymerases in Yeast

“…Gpn3 has been proposed to mediate RNA pols biogenesis, assembly, and transport to the nucleus ( Minaker et al, 2013 ; Liu et al, 2020 ) and has been demonstrated to form a stable complex with Gpn1 in both yeast and human ( Carre and Shiekhattar, 2011 ; Cristóbal-Mondragón et al, 2019 ; Liu et al, 2020 ). In addition, yeast gpn3 mutants mislocalize RNA pol II and III subunits, which suggests a role for Gpn3 (and Gpn2) in not only RNA pol II but also in RNA pol III assembly and/or transport ( Minaker et al, 2013 ).…”

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 .…”

“…14 Here, we clearly showed that Gpn3 and Npa3 in yeast are mutually required to stabilize their protein levels ( Figure 4D,E), and that Gpn3 coordinates with Npa3 for RNAPII biogenesis. Although the association of Gpn1 with Gpn3 have been reported in human cells, 21,23 the specific role of Gpn3 in the biogenesis of RNAPII is not quite clear. Depletion of either Npa3 or Gpn3 leads to severe growth defects ( Figure 1A) indicates they are not redundant during RNAPII assembly.…”

“…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.…”