Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus

Abstract

A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nucla gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (~ 190 kD and 2 x 104 copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nucl+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.