RESUMEN
The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.
Asunto(s)
Factor 1 Eucariótico de Iniciación , Higromicina B , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Núcleo Celular/metabolismo , Núcleo Celular/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Higromicina B/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismoRESUMEN
Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting.
Asunto(s)
Neoplasias de la Mama/enzimología , Núcleo Celular/enzimología , Codón sin Sentido , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Polimerasa II/metabolismo , Transporte Activo de Núcleo Celular/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/genética , Citoplasma/enzimología , Citoplasma/genética , Femenino , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Dominios PDZ , ARN Polimerasa II/genéticaRESUMEN
The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.
Asunto(s)
Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/aislamiento & purificación , Guanosina Trifosfato/química , Multimerización de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión al GTP/genética , Humanos , Hidrólisis , Dominios Proteicos , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.
Asunto(s)
Núcleo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Microtúbulos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Benomilo/farmacología , Viabilidad Microbiana , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas de Unión al GTP Monoméricas/metabolismo , Dominios Proteicos , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Moduladores de Tubulina/farmacología , Vacuolas/metabolismoRESUMEN
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.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Unión ProteicaRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , GTP Fosfohidrolasas/fisiología , ARN Polimerasa II/metabolismo , Mama/metabolismo , Núcleo Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , GTP Fosfohidrolasas/antagonistas & inhibidores , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Inmunoprecipitación , Unión Proteica , Transporte de Proteínas/genética , Interferencia de ARN/fisiología , ARN Interferente Pequeño/farmacología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
DNA damage is at the center of the genesis, progression and treatment of cancer. We review here the molecular mechanisms of the DNA damage inducing small molecules most commonly used in cancer therapy. Cell cycle control and DNA repair mechanisms are known to be activated after DNA damage. Here, we revise recent discoveries related to the cell cycle control and DNA repair processes and how these findings are being utilized for the more efficient, powerful and selective therapies for cancer treatment.