Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.

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.

[1]  A. Newman.  Pre-mRNA splicing. , 1994, Current Opinion in Genetics and Development.

[2]  M. Snyder,et al.  The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton , 1992, The Journal of cell biology.

[3]  L. Maquat.  Nuclear mRNA export. , 1991, Current opinion in cell biology.

[4]  D. Spector,et al.  Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. , 1991, Genes & development.

[5]  W. Ellmeier,et al.  Mature mRNA 3′ end formation stimulates RNA export from the nucleus. , 1991, The EMBO journal.

[6]  D. Kipling,et al.  Recombination and RNA processing: a common strand? , 1991, Trends in cell biology.

[7]  M. Schmidt,et al.  Requirement for acidic amino acid residues immediately N‐terminal to the conserved domain of Saccharomyces cerevisiae TFIID. , 1991, The EMBO journal.

[8]  J. Hesketh,et al.  Interaction between mRNA, ribosomes and the cytoskeleton. , 1991, The Biochemical journal.

[9]  D. Kipling,et al.  rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins. , 1991, Nucleic acids research.

[10]  J. Lawrence,et al.  Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation , 1991, The Journal of cell biology.

[11]  S. Ruby,et al.  Pre-mRNA splicing in yeast. , 1991, Trends in genetics : TIG.

[12]  S. Lindquist,et al.  Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae , 1991, Molecular and cellular biology.

[13]  J. Keene,et al.  Nuclear RNA-binding proteins. , 1991, Progress in nucleic acid research and molecular biology.

[14]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[15]  G. Fink,et al.  kem mutations affect nuclear fusion in Saccharomyces cerevisiae. , 1990, Genetics.

[16]  P. Agutter.  Between Nucleus and Cytoplasm , 1990 .

[17]  C. Thummel,et al.  Splicing precedes polyadenylation during Drosophila E74A transcription , 1990, Molecular and cellular biology.

[18]  F. Larimer,et al.  Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth. , 1990, Gene.

[19]  I. Mattaj,et al.  Monomethylated cap structures facilitate RNA export from the nucleus , 1990, Cell.

[20]  A. Hopper,et al.  The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus , 1990, The Journal of cell biology.

[21]  J. Verdier.  Regulatory DNA‐binding proteins in yeast: An overview , 1990, Yeast.

[22]  S. Gasser,et al.  The composition and morphology of yeast nuclear scaffolds. , 1990, Journal of cell science.

[23]  H. Horstmann,et al.  NSP1: A yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain , 1990, Cell.

[24]  T. A. Brown,et al.  A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. , 1990, Nucleic acids research.

[25]  E. Buhle,et al.  Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components , 1990, The Journal of cell biology.

[26]  J. Nickerson,et al.  Core filaments of the nuclear matrix , 1990, The Journal of cell biology.

[27]  A. Cigan,et al.  Sequence and structural requirements for efficient translation in yeast. , 1990, Methods in enzymology.

[28]  Y. Shimura,et al.  A nuclear cap binding protein from HeLa cells. , 1990, Nucleic acids research.

[29]  K. McEntee,et al.  Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae. , 1989, The Journal of biological chemistry.

[30]  P. Sharp,et al.  Regulation by HIV Rev depends upon recognition of splice sites , 1989, Cell.

[31]  M. Douglas,et al.  Organization of the nuclear pore complex in Saccharomyces cerevisiae. , 1989, Journal of ultrastructure and molecular structure research.

[32]  G. Blobel,et al.  Primary structure analysis of an integral membrane glycoprotein of the nuclear pore , 1989, The Journal of cell biology.

[33]  P. Legrain,et al.  Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm , 1989, Cell.

[34]  Robert H. Singer,et al.  Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization , 1989, Cell.

[35]  T. Copeland,et al.  rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Zeitlin,et al.  Autonomous splicing and complementation of in vivo-assembled spliceosomes , 1989, The Journal of cell biology.

[37]  J. Butler,et al.  RNA processing generates the mature 3' end of yeast CYC1 messenger RNA in vitro. , 1988, Science.

[38]  D. Jackson,et al.  Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat. , 1988, The EMBO journal.

[39]  N. Proudfoot,et al.  A beginning to the biochemistry of polyadenylation. , 1988, Trends in genetics : TIG.

[40]  J. Knowland,et al.  Attachment of transcriptionally active DNA sequences to the nucleoskeleton under isotonic conditions. , 1988, Nucleic acids research.

[41]  A. Sluder,et al.  Elongation by Drosophila RNA polymerase II. Transcription of 3'-extended DNA templates. , 1988, The Journal of biological chemistry.

[42]  Y. Osheim,et al.  Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. , 1988, Genes & development.

[43]  C. Kane.  Renaturase and ribonuclease H: a novel mechanism that influences transcript displacement by RNA polymerase II in vitro. , 1988, Biochemistry.

[44]  W. V. van Venrooij,et al.  The nuclear matrix: structure and composition. , 1988, Journal of cell science.

[45]  C. Feldherr,et al.  Translocation of RNA-coated gold particles through the nuclear pores of oocytes , 1988, The Journal of cell biology.

[46]  R. Young,et al.  Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis , 1987, Molecular and cellular biology.

[47]  A. Stevens,et al.  A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity. , 1987, Archives of biochemistry and biophysics.

[48]  S. Zeitlin,et al.  Pre-mRNA splicing and the nuclear matrix , 1987, Molecular and cellular biology.

[49]  H. Schröder,et al.  Transport of mRNA from nucleus to cytoplasm. , 1987, Progress in nucleic acid research and molecular biology.

[50]  D. Botstein,et al.  A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. , 1987, Gene.

[51]  A. Bankier,et al.  Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. , 1987, Methods in enzymology.

[52]  G. Natsoulis,et al.  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. , 1987, Methods in enzymology.

[53]  G. Blobel,et al.  Identification and characterization of a nuclear pore complex protein , 1986, Cell.

[54]  S. Leff,et al.  Complex transcriptional units: diversity in gene expression by alternative RNA processing. , 1986, Annual review of biochemistry.

[55]  G. Dreyfuss.  Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. , 1986, Annual review of cell biology.

[56]  A. Hopper,et al.  Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype , 1985, Molecular and cellular biology.

[57]  B. Vogelstein,et al.  The association of transcribed genes with the nuclear matrix of Drosophila cells during heat shock. , 1985, Nucleic acids research.

[58]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[59]  P. Hentzen,et al.  Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[60]  B. O’Malley,et al.  Actively transcribed genes are associated with the nuclear matrix , 1983, Nature.

[61]  F. Sherman,et al.  DNA sequence required for efficient transcription termination in yeast , 1982, Cell.

[62]  B. Vogelstein,et al.  The ovalbumin gene is associated with the nuclear matrix of chicken oviduct cells , 1982, Cell.

[63]  C. Chae,et al.  Association of globin ribonucleic acid and its precursors with the chicken erythroblast nuclear matrix. , 1982, Biochemistry.

[64]  M. J. Johnson,et al.  A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322. , 1981, Gene.

[65]  M. Rosbash,et al.  The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast , 1981, Cell.

[66]  R. Mortimer,et al.  Genetic map of Saccharomyces cerevisiae. , 1980, Microbiological reviews.

[67]  A. Banerjee,et al.  5'-terminal cap structure in eucaryotic messenger ribonucleic acids , 1980 .

[68]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[69]  A. Hopper,et al.  A yeast mutant which accumulates precursor tRNAs , 1978, Cell.

[70]  J. Garnier,et al.  Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. , 1978, Journal of molecular biology.

[71]  P. Y. Chou,et al.  Empirical predictions of protein conformation. , 1978, Annual review of biochemistry.

[72]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[73]  B. Groner,et al.  Polyadenylate metabolism in the nuclei and cytoplasm of Saccharomyces cerevisiae. , 1975, The Journal of biological chemistry.

[74]  K. Shiokawa,et al.  The role of cytoplasmic membranes in controlling the transport of nuclear messenger RNA and initiation of protein synthesis. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[75]  H. Swift,et al.  RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS , 1966, The Journal of cell biology.