Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes 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 nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains 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 describe here the properties of yeast strains bearing the rat3-1 mutation (RAT-ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37 degrees C for 4 h. In wild-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 were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of 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 periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.
Li
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Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Heath
C V
CV
Amberg
D C
DC
Dockendorff
T C
TC
Copeland
C S
CS
Snyder
M
M
Cole
C N
CN
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics; D002874:Chromosome Mapping; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000502:physiology; D017404:In Situ Hybridization, Fluorescence; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009685:Nuclear Envelope / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; C091271:NUP133 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins