• Abstract. To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A) ÷ RNA in their nuclei when shifted to the non-permissive temperature of 37°C. We describe here the properties of yeast strains carrying mutations in the RA T2 gene (RA Tribonucleic acid trafficking) and the cloning of the RA T2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A) + RNA when cultured at 15 ° or 23°C, but within 4 h of a shift to the nonpermissive temperature of 37°C, poly(A) ÷ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope 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 mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RA T2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37°C, but the growth defect at high temperature could be suppressed by growth of mutant ceils in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaC1. The phenotypes seen in cells carrying a disruption of the RA T2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7-1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2pfNupl20p.

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rsslp encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rsslp partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37°C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37°C, the mutant Rat7-lp/Nupl59-lp is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rsslp did not result in retention of the mutant Rat7-lp/Nupl59-lp in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rsslp by placing the RSS1 open reading frame (ORF) under control of the GALl promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rsslp are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rsslp overexpression suppresses the growth defect of rat7-1 cells at 37°C by acting to maintain mRNA export.

Rat7p/Nup159p is an essential nucleoporin of Sac-charomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export. Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 100 amino acids upstream of the actual stop codon of this 1,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein. These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions. In contrast, no other region of the protein was absolutely required. The SAFG/PSFG repeat region in the central third of the protein was completely dispensable for growth at temperatures between 16 degrees C and 37 degrees C and cells expressing this mutant allele were indistinguishable from wild type. Deletion of the amino-terminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs). Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences. In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.