MUTATION OR DELETION OF THE SACCHAROMYCES-CEREVISIAE RAT3 NUP133 GENECAUSES TEMPERATURE-DEPENDENT NUCLEAR ACCUMULATION OF POLY(A)(+) RNA AND CONSTITUTIVE CLUSTERING OF NUCLEAR-PORE COMPLEXES/

To identify genes whose products play potential roles in the nucleocyt oplasmic export of messenger RNA, we isolated temperature-sensitive st rains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)(50) probe, we identified those that showed nuclear accumulation of poly(A) (+) RNA when cells were shifted to the nonpermissive temperature. We d escribe here the properties of yeast strains bearing the rat3-1 mutati on (RAT - ribonucleic acid trafficking) and the cloning of the RAT3 ge ne. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulat ion of poly(A)(+) RNA, whereas approximately 70% showed nuclear accumu lation of poly(A)(+) RNA after a shift to 37 degrees C for 4 h. In wil d-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs w ere clustered into one or a few regions of the NE in mutant cells. Sim ilar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-a mino acid protein without similarity to other known proteins. It is es sential for growth only at 37 degrees C. Cells carrying a disruption o f the RAT3 gene were very similar to cells carrying the original rat3- 1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)(+) RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periph ery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.