Isolation and characterization of the RNA2+, RNA4+, and RNA11+ genes of Saccharomyces cerevisiae

Soltyk, A; Tropak, M; Friesen, James D.

doi:10.1128/jb.160.3.1093-1100.1984

Cited by 28 publications

(12 citation statements)

References 56 publications

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“…The anti-RNA2 sera identified a polypeptide of 95-100 kD as the RNA2 polypeptide. This molecular mass is consistent with the 2,500-3,000 nt size of the mRNA reported in the literature (Last et al, 1984;Lee et al, 1984;Soltyk et al, 1984). Lee et al (manuscript submitted for publication) have identified a protein of 97-105 kD using either antisera directed against a peptide from the predicted RNA2 amino terminus, or antisera raised against fusion protein containing 151 amino acids from the middle third of the predicted RNA2 ORE Our R N A 2 0 R F 1 fusion protein contains a region of the RNA2 protein distinct from those used by Lee et al (J. Beggs, personal communication).…”

Section: Discussionsupporting

confidence: 91%

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Woolford

1986

The Journal of Cell Biology

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Abstract. Temperature-sensitive mutations in the RNA2 through RNA/I genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.M UCH of the interest in expression of RNA polymerase lI transcribed genes is focused on defining the mechanism of processing the primary transcription products of these genes (pre-mRNAs) to cytoplasmic, translatable mRNAs. Accurate splicing of introns from pre-mRNAs is absolutely required for production of functional polypeptides from mosaic genes. The modulation of pre-mRNA processing patterns to generate polypeptide diversity during development of eukaryotic viruses (Nevins, 1982) and metazoan organisms (DeNoto et al., 1981; Schwarzbauer et al., 1983; Periasamy et al., 1985) is widespread, and elucidation of its molecular mechanisms is of great importance. This has become possible since the development of well-defined soluble in vitro systems that are capable of processing intron-containing exogenously added pre-mRNA molecules, using mammalian whole-ceil and nuclear extracts (Frendewey and Keller, 1985;Grabowski et al., 1985;Krainer and Maniatis, 1985) or yeast cell extracts (Brody and Abelson, 1985;Lin et al., 1985). Unique species with kinetic properties consistent with pre-mRNA processing intermediates have been identified in in vitro processing reactions Ruskin et al., 1984;Lin et al., 1985). The in vitro pre-mRNA processing mechanism appears to mimic reactions occurring in vivo, since similar intermediates have also been detected in mammalian cells (Wallace and Edmonds, 1983;Zeitlin and Efstratiadis, 1984) and yeast (Domdey et al., 1984;Rodriguez et al., 1984).Although the budding yeast Saccharomyces cerevisiae has a relatively small number of intron-containing genes (Gallwitz and Seidel, 1980;Ng and Abelson, 1980; Rosbash et al., 1981;Fried et al., 1981;Larkin and Woolford, 1983;Miller, 1984), the powerful molecular-genetic techniques available in this organism (Botstein and Davis, 1982), and the rapidly advancing cell biological technology make this an excellent system for studying pre-mRNA processing both in vivo and in vitro. The availability of a yeast in vitro splicing system (Brody and Abelson, 1985;Lin et al., 1985) provides the opportunity to analyze the functions of purified proteins in the pre-mRNA processing reaction.A unique advantage of studying pre-mRNA processing in yeast is the existence of 10 complementation groups of temperature-sensitive mutants, rna2 through rna// (Hartwell, 1967), that have been shown to be defective in premRNA processing (Rosba...

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Section: Discussionsupporting

confidence: 91%

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Woolford

1986

The Journal of Cell Biology

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“…Ten of these mutants, rna2 to rnal 1, originally described as defective in proper ribosome formation or maturation (Hartwell et al, 1970), were shown to accumulate unspliced pre-mRNAs at the nonpermissive temperature (Fried et al, 1981;Rosbash et al, 1981;Teem and Rosbash, 1983). Cloning of the corresponding genes (Last et al, 1984;Lee et al, 1984;Soltyk et al, 1984;Jackson et al, 1988;Abovitch et al, 1990) and analysis of splicing extracts prepared from rna2, 3, 4, 7, 8, and 11 cells for heat lability (Lustig et al, 1986) revealed that proteins encoded by these RNA genes indeed participate in the excision of intervening sequences from mRNA precursors. Therefore, the RNA2-11 genes are now referred to as PRP (for pre-mRNA processing).…”

Section: Introductionmentioning

confidence: 99%

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

Melchior

Weber

Gerke

1993

MBoC

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The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.

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“…The mutations also highlighted genes involved in pre-mRNA splicing. Some of these genes have been cloned (Last et al, 1984;Lee et al, 1984;Soltyk et al, 1984;Jackson et al, 1988) and have been shown to encode proteins (Last and Woolford, 1986; Lee et al, 1986;Jackson et al, 1988). The RNA2 and RNA3 gene products are found within the nucleus (Last and Woolford, 1986) and the RNAlJ protein has been localized to the periphery of the nucleus (Chang et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

Potashkin

Frendewey

1989

The EMBO Journal

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A collection of temperature sensitive (ts‐) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts‐ mutants for pre‐mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta‐tubulin pre‐mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre‐mRNA splicing mutants from 100 ts‐ strains. The three mutants are defective in an early step of the pre‐mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre‐mRNA showed that the mutations are not specific for beta‐tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre‐tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts‐ growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.

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Isolation and characterization of the RNA2+, RNA4+, and RNA11+ genes of Saccharomyces cerevisiae

Cited by 28 publications

References 56 publications

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

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