Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

Abstract

Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1⌬ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1⌬ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.A ll eukaryotic cells have at least three different RNA polymerases (Pol). Pol I synthesizes the large precursor of rRNA, Pol II produces mainly mRNAs, and Pol III generates tRNAs, 5S rRNA and other small noncoding RNAs.Pol III, which is the focus of this work, is a heteromultimeric protein complex that contains 17 distinct subunits in Saccharomyces cerevisiae. The two largest subunits, C160 and C128, form opposite sides of the active-center cleft and harbor the catalytic activity, and they are related to the ␤= and ␤ components of the ␣ 2 ␤␤= core of bacterial RNA polymerase. Homology to the bacterial ␣ subunit, although less strong, was also observed, with the subunit AC40 common for yeast and Pol III and Pol I. A second ␣-like subunit, AC19, forms a heterodimer with AC40, which is a functional equivalent to the ␣ 2 homodimer in prokaryotes (1). The polymerase core, in addition to the ␣ 2 ␤␤=-like structure, contains six small subunits, which are structurally and functionally conserved from yeast to humans. The small core subunits either bind or bridge the two largest catalytic subunits. In addition to the central core, the remaining subunits of Pol III are organized in heterodimeric or trimeric stalks or subdomains. Significantly, the C37 to C53 and C82 to C34 subdomains, which are stably bound to the RNA Pol III core, resemble Pol II general transcription factors TFIIF and TFIIE (2).Little is known about how polymerase complexes are assembled from the subunits, how they reach their functional destination, and how they dissociate after transcription. In bacteria, the assembly of RNA polymerase starts with the formation of the ␣␣ dimer, which then interacts with the ␤ subunit to yield an ␣␣␤...

Coupling of RNA polymerase III assembly to cell cycle progression in Saccharomyces cerevisiae

“…This outcome also indicates that the effect of increased RBS1 expression on the cell cycle is specific to rpc128-1007-mediated Pol III assembly defects. This result is consistent with an earlier study showing that rpc128-1007 was the only one among several other Pol III mutants tested for which the growth phenotype could be suppressed by RBS1 overexpression [10].…”

“…We have recently described an yeast Pol III mutation, rpc128-1007, which occurs in the C-terminal region of second largest subunit of Pol III, Rpc128. This mutant strain has a severe defect in Pol III complex assembly in addition to the previously documented reduction in tRNA levels [10,11]. Genetic suppressor screening for genes that can suppress the cold-sensitive growth phenotype of the rpc128-1007 mutant identified the gene encoding the ABC10β subunit that is shared by all three RNA polymerases and is involved in polymerase assembly [12].…”

“…Rbs1 also interacts with the exportin Crm1 and shuttles between the cytoplasm and the nucleus. We thus postulated that Rbs1 protein functions as an assembly/import factor for the Pol III complex [10]. Numerous previous studies concerning the biogenesis of multi-subunit RNA polymerases suggest that the Pol III complex is assembled in the cytoplasm with the help of assembly factors and then transported to the nucleus where it transcribes tRNA genes (reviewed in [13,14]).…”

“…Genetic suppressor screening for genes that can suppress the cold-sensitive growth phenotype of the rpc128-1007 mutant identified the gene encoding the ABC10β subunit that is shared by all three RNA polymerases and is involved in polymerase assembly [12]. Pol III assembly defect and cold-sensitive growth phenotype were also suppressed by overproduction of Rbs1, which physically interacts with a subset of Pol III subunits: AC19, AC40 and ABC27/Rpb5 [10]. Rbs1 also interacts with the exportin Crm1 and shuttles between the cytoplasm and the nucleus.…”

Maf1 phenotypes and cell physiology

“…The position of this mutation in the structure of Pol III suggests a specific assembly/stability defect. Consistent with this, FBA1 overexpression did not suppress conditional phenotypes resulting from mutations in other Pol III subunits (Rpc160, Rpc31 and Rpc11) and Pol III subunit abundance was significantly reduced in whole cell extracts and in Pol III immunoprecipitates from the Rpc128 mutant strain [31]. FBA1 suppression of the rpc128-1007 cold-sensitive growth phenotype increased the low level of precursor and mature tRNAs in these cells.…”