RESUMEN
Although remarkable progress has been made toward understanding the structures of eukaryotic RNA polymerases, the pathways and factors that facilitate their assembly remain unresolved. Essential proteins Rba50 and Gpn2 are required for Rpb3 subcomplex assembly, but whether they participate in subsequent assembly steps is unknown. Herein, we performed comprehensive genetic screens to explore Rba50 function. We identified two unique extragenic rba50-3-suppressing mutations that map to genes encoding the Rba50-interacting protein Gpn2, and Rpb2, the second largest subunit of RNAPII. Both gpn2-R347S and rpb2-V1171G variants bypass Rpb1 cytoplasmic arrest and temperature-sensitive growth defects of the rba50-3 mutant. GPN2 and RPB2 were also identified as novel multicopy suppressors of the rba50-3 mutant. Rapid depletion of Rba50 affected Rpb3-Rpb2 association during RNAPII assembly. Importantly, we demonstrated that Gpn2 facilitates the association of Rba50 and Rpb2. Our results imply that Rba50-Gpn2 interaction is essential for Rpb2 recruitment during RNAPII assembly following Rpb3 subcomplex assembly. Furthermore, the Rba50-Gpn2 complex appears to play a similar role in the assembly of RNAPIII. We therefore propose a model in which Rba50 interacts with Gpn2 and thereby promotes loading of the second largest subunit of RNAP II and III onto the previously assembled subcomplex.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , ARN Polimerasa II , Citoplasma/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Mutación , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismoRESUMEN
RNA polymerase II is one of the most vital macromolecular complexes in eukaryotes and the assembly of such complete enzyme requires many factors. Three members of GPN-loop GTPase family Npa3/Gpn1, Gpn2, and Gpn3 participate in the biogenesis of RNA polymerase II with nonredundant roles. We show here that rapid degradation of each GPN protein in yeast leads to cytoplasmic accumulation of Rpb1 and defects in the assembly of RNA polymerase II, suggesting conserved functions of GPN paralogs for RNA polymerase II biogenesis as in humans. Taking advantage of a multicopy genetic screening, we identified GPN3 and assembly factor RBA50 among others as strong suppressors of npa3ts mutants. We further demonstrated that Npa3 interacts with Gpn3 and Rba50, similarly human Gpn1 physically interacts with Gpn3 and RPAP1 (human analog of Rba50). Moreover, a mutual dependency of protein levels of Npa3 and Gpn3 was also clearly presented in yeast using an auxin-inducible degron (AID) system. Interestingly, Rpb2, the second largest subunit of RNA polymerase II was determined to be the subunit that interacts with both Gpn1 and Rba50, indicating a close association of Npa3 and Rba50 in Rpb2 subcomplex assembly. Based on these results, we conclude that Npa3 interacts with Gpn3 and Rba50, for RNA polymerase II biogenesis. We therefore propose that multiple factors may coordinate through conserved regulatory mechanisms in the assembly of RNA polymerase complex.
Asunto(s)
Proteínas Portadoras/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Unión Proteica , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of 12 subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, and thus the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, that directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. An interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 subcomplex is disrupted, leading to defects in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 subcomplex and subsequently the biogenesis of RNAPII.