title: Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.
abstract: We have combined techniques of genetics and histochemistry to identify genes required for the nucleocytoplasmic export of mRNA in the budding yeast Saccharomyces cerevisiae. We adapted in situ hybridization using a digoxigenin-labeled oligo(dT)50 probe to localize poly(A)+ RNA in fixed yeast cells and used yeast strains carrying the rna1-1 mutation to develop an assay. The rna1-1 mutation is the only previously described mutation that causes defects in mRNA export. As visualized with this RNA localization assay, rna1-1 strains accumulated poly(A)+ RNA at the nuclear periphery at the nonpermissive temperature. This was in contrast to the RNA localization pattern of wild-type cells or rna1-1 cells grown at permissive temperature. Wild-type cells showed bright uniform cytoplasmic staining with little detectable RNA in the nuclei. We used this RNA localization assay to screen a bank of temperature-sensitive yeast strains for mutants with inducible defects in mRNA trafficking. Strains identified in this manner are designated RAT mutants for ribonucleic acid trafficking. The rat1-1 allele conferred temperature-sensitive accumulation of poly(A)+ RNA in one to several intranuclear spots that appear to lie at the nuclear periphery. RNA processing was unaffected in rat1-1 strains, except for an inducible defect in trimming the 5' end of the 5.8S rRNA. The wild-type RAT1 gene was cloned by complementation; it encodes an essential 116-kD protein with regions of homology to the protein encoded by SEP1 (also known as DST2, XRN1, KEM1, and RAR5). Sep1p is a nucleic acid binding protein, a 5'----3' exonuclease, and catalyzes DNA strand transfer reactions in vitro. We discuss the possible significance of the Rat1p/Sep1p homology for RNA trafficking. We also discuss the potential of this RNA localization assay to identify genes involved in nuclear structure and RNA metabolism.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755.
authors.forename: D C
authors.initials: DC
authors.lastname: Amberg
authors.forename: A L
authors.initials: AL
authors.lastname: Goldstein
authors.forename: C N
authors.initials: CN
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004271:DNA, Fungal; D005095:Exoribonucleases; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D012331:RNA, Fungal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C075577:RAT1 protein, S cerevisiae; D005095:Exoribonucleases