Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene.

Abstract

• 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.

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

“…In nup159 cells, the phenomenon is actually the opposite of that in gle2 cells: nup159 NPCs are clustered at 23°C, whereas at 37°C their distribution is over the entire nuclear surface (Gorsch et al, 1995). The herniated clustered NPCs in gle2 cells are distinct from the successive interconnected NPCs found in grape-like aggregates of constitutively clustered NPCs in nup145, nup120, nup84, and nup85 mutant cells (Wente and Blobel, 1994;Aitchison et al, 1995a;Heath et al, 1995;Goldstein et al, 1996;Siniossoglou et al, 1996). Cells deleted for NUP116 form herniations when shifted to the restrictive temperature (Wente and Blobel, 1993 (Wente and Blobel, 1993).…”

“…Thus, protein import was not noticeably diminished. This suggested that Gle2p is specifically required for a NPC export func- (Wente and Blobel, 1993;Bogerd et al, 1994;Doye et al, 1994;Aitchison et al, 1995a;Gorsch et al, 1995;Heath et al, 1995;Li et al, 1995;Goldstein et al, 1996;Siniossoglou et al, 1996).…”

“…Mutations in NUP145 (Wente and Blobel, 1994), NUP133 (Doye et al, 1994;Li et al, 1995;Pemberton et al, 1995), NUP120 (Aitchison et al, 1995a;Heath et al, 1995), NUP159 (Gorsch et al, 1995), NUP84 (Siniossoglou et al, 1996), and NUP85 (Goldstein et al, 1996;Siniossoglou et al, 1996) (Figure 6, right). The same was true for gle2 cells grown at 23°C (igure 6, top left).…”

“…Collections of temperaturesensitive mutant yeast strains have been directly screened for blocks in polyadenylated poly(A)+ RNA export (Amberg et al, 1992;Kadowaki et al, 1992Kadowaki et al, , 1994. Interestingly, numerous rat mutants isolated by Cole and coworkers encode for nucleoporins (rat2/ nup120, rat3/nupl33, rat7/nupl59, and rat9/nup85; Gorsch et al, 1995;Heath et al, 1995;Li et al, 1995;Goldstein et al, 1996) that may play a direct role in export.…”

Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex

“…The deletion or immunodepletion of any nup from these complexes has dramatic consequences on the architecture and function of the NPC, as well as on the organization of the nuclear envelope (17,20,(23)(24)(25)(26). In particular, Nup120 is involved in nuclear poly(A) ϩ mRNA and preribosome export, NPC assembly and distribution, and nuclear envelope organization (26)(27)(28).…”