Parcs/Gpn3 is a putative GTPase that is conserved in eukaryotic cells from yeast to humans, suggesting that it plays a fundamental, but still unknown, cellular function. Suppression of Parcs/Gpn3 expression by RNAi completely blocked cell proliferation in MCF-12A cells and other mammary epithelial cell lines. Unexpectedly, Parcs/Gpn3 knockdown had a more modest effect in the proliferation of the tumorigenic MDA-MB-231 and SK-BR3 cells. RNA polymerase II (RNAP II) co-immunoprecipitated with Parcs/Gpn3. Parcs/Gpn3 depletion caused a reduction in overall RNA synthesis in MCF-12A cells but not in MDA-MB-231 cells, demonstrating a role for Parcs/Gpn3 in transcription, and pointing to a defect in RNA synthesis by RNAP II as the possible cause of halted proliferation. The absence of Parcs/Gpn3 in MCF-12A cells caused a dramatic change in the sub-cellular localization of Rpb1, the largest subunit of RNAP II. As expected, Rpb1 was present only in the nucleus of MCF-12A control cells, whereas in Parcs/Gpn3-depleted MCF-12A cells, Rpb1 was detected exclusively in the cytoplasm. This effect was specific, as histones remained nuclear independently of Parcs/Gpn3. Rpb1 protein levels were markedly increased in Parcs/Gpn3-depleted MCF-12A cells. Interestingly, Rpb1 distribution was only marginally affected after knocking-down Parcs/Gpn3 in MDA-MB-231 cells. In conclusion, we report here, for the first time, that Parcs/Gpn3 plays a critical role in the nuclear accumulation of RNAP II, and we propose that this function explains the relative importance of Parcs/Gpn3 in cell proliferation. Intriguingly, at least some tumorigenic mammary cells have evolved mechanisms that allow them to proliferate in a Parcs/Gpn3-independent manner.
Edited by Ivan SadowskiKeywords: Gpn1 Gpn3 Gpn1-Gpn3 interaction Gpn1-Gpn3 nucleocytoplasmic shuttling Interdependent protein levels shRNA a b s t r a c t Gpn1 and Gpn3 are GTPases individually required for nuclear targeting of RNA polymerase II. Here we show that whereas Gpn3-EYFP distributed between the cytoplasm and cell nucleus, it was mainly cytoplasmic when coexpressed with Gpn1-Flag. Gpn3-Flag retained Gpn1-EYFP in the cytoplasm. However, Gpn3-EYFP/Gpn1-Flag nucleocytoplasmic shuttling was revealed after inhibiting nuclear export with leptomycin B. All Gpn3-EYFP coimmunoprecipitated with Gpn1-Flag, and all Gpn1-EYFP with Gpn3-Flag. Importantly, most endogenous Gpn1 and Gpn3 also associate. Gpn1-Gpn3 interaction was essential to maintain steady-state protein levels of both GTPases. We propose that most Gpn1 and Gpn3 associate, are mobilized, and function as a protein complex.
Structured summary of protein interactions:GPN3 physically interacts with GPN1 by anti tag coimmunoprecipitation (1, 2) GPN3 and GPN1 colocalize by fluorescence microscopy (View interaction)
XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.