NUCLEAR-PORE COMPLEX CLUSTERING AND NUCLEAR ACCUMULATION OF POLY(A)(-CEREVISIAE RAT2() RNA ASSOCIATED WITH MUTATION OF THE SACCHAROMYCES)NUP120 GENE/

To identify genes involved in the export of messenger RNA from the nuc leus to the cytoplasm, we used an in situ hybridization assay to scree n temperature-sensitive strains of Saccharomyces cerevisiae. This iden tified those which accumulated poly(A)(+) RNA in their nuclei when shi fted to the non-permissive temperature of 37 degrees C. We describe he re the properties of yeast strains carrying mutations in the RAT2 gene (RAT-ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nucle ar accumulation of poly(A)(+) RNA when cultured at 15 degrees or 23 de grees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)(+) RNA accumulated within the nuclei of approxi mately 80% of cells. No defect was seen in the nuclear import of a rep orter protein bearing a nuclear localization signal, Nuclear pore comp lexes (NPCs) are distributed relatively evenly around the nuclear enve lope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope, This clustering was a constitutive property of muta nt cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute ar ound the nuclear envelope when nuclei are separated from cytoplasmic c omponents. Electron microscopy revealed that these clusters were frequ ently found in a protuberance of the nuclear envelope and were often l ocated close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential f or growth only at 37 degrees C, but the growth defect at high temperat ure could be suppressed by growth of mutant cells in the presence of h igh osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phen otypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagg ing was used to show that Rat2p is located at the nuclear periphery an d co-localizes with yeast NPC proteins recognized by the RL1 monoclona l antibody. The rat2-1 allele was synthetically lethal with both the r at3-1/nup133-1 and rat7-1/nup159-1 alleles. These results indicate tha t the product of this gene is a nucleoporin which we refer to as Rat2p /Nup120p.