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C Guthrie

Publications and source records attributed to C Guthrie.

At least 91 records · Page 5Linked to original sources
PubMed1987

Saccharomyces cerevisiae has a U1-like small nuclear RNA with unexpected properties.

Previous experiments indicated that only a small subset of the approximately equal to 24 small nuclear RNAs (snRNAs) in Saccharomyces cerevisiae have binding sites for the Sm antigen, a hallmark of metazoan small nuclear ribonucleoproteins (snRNPs) involved in pre-messenger RNA splicing. Antibodies from human serum to Sm proteins were used to show that four snRNAs (snR7, snR14, snR19, and snR20) can be immunoprecipitated from yeast extracts. Three of these four, snR7, snR14, and snR20, have been shown to be analogs of mammalian U5, U4, and U2, respectively. Several regions of significant homology to U1 (164 nucleotides) have now been found in cloned and sequenced snR19 (568 nucleotides). These include ten out of ten matches to the 5' end of U1, the site known to interact with the 5' splice site of mammalian introns. Surprisingly, the precise conservation of this sequence precludes perfect complementarity between snR19 and the invariant yeast 5' junction (GTATGT), which differs from the mammalian consensus at the fourth position (GTPuAGT).

Animals↗
PubMed1987

An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA.

Three yeast snRNAs (snR20, snR7, and snR14) have been implicated in pre-mRNA splicing. snR20 and snR7 contain domains of homology to U2 and U5, respectively, and each is required for viability. These RNAs are found associated with the spliceosome, as is snR14. We show here that snR14 is also an essential gene product. Sequence analysis reveals that, like snR7 and snR20, snR14 contains a consensus binding site for the Sm antigen, a feature common to all mammalian snRNAs involved in splicing. Moreover, snR14 exhibits several blocks of sequence and structural homology to U4, which in metazoans is found in association with U6. Native gel electrophoresis demonstrates that snR14 is in fact base-paired with another yeast snRNA, designated snR6, which has primary sequence homology to U6. We conclude that snR14 is the yeast analog of U4.

Animal Population Groups↗
PubMed1987

An essential yeast snRNA with a U5-like domain is required for splicing in vivo.

Yeast contains at least 24 snRNAs, many of which are dispensable for viability. We recently demonstrated that a small subset of these RNAs has a functional binding site for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. Here we show that one of these snRNAs, snR7, is required for growth. To determine the biochemical basis of lethality in cells lacking snR7, we engineered the conditional synthesis of snR7 by fusing the snRNA coding sequences to the yeast GAL1 control region. Cells depleted for the SNR7 gene product by growth on glucose for five generations show marked accumulation of unspliced mRNA precursors from the four intron-containing genes tested. In some cases, intron-exon 2 lariats also accumulate. We have identified a 70 nucleotide domain within snR7 with limited sequence-specific but striking structural homology to the mammalian snRNA U5. We conclude that mRNA splicing in yeast requires the function of a U5-like snRNA.

Base Sequence↗
PubMed1987

Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA.

The U2 snRNP binds to the site of branch formation during splicing of mammalian pre-mRNA in vitro. In Saccharomyces cerevisiae the branch site is within the so-called TACTAAC box (UACUAAC box), an absolutely conserved intron sequence required for splicing. Based on the identification and sequence of a U2 analogue in yeast, a specific base pairing interaction between the UACUAAC box and a highly conserved region of this snRNA can be proposed. To test this hypothesis, we have taken advantage of two mutations constructed previously in the UACUAAC box of an actin-HIS4 fusion. These mutant strains were transformed with stable plasmids bearing U2-like snRNAs into which changes predicted to restore base pairing had been introduced. Allele-specific suppression of biological and biochemical phenotypes was observed in both cases. Recognition of the UACUAAC box thus relies, at least in part, on Watson-Crick base pairing with the yeast U2 analogue.

Alleles↗
PubMed1987

A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen.

Autoimmune sera of the Sm specificity react with the major class of small nuclear RNA (snRNA)-containing ribonucleoprotein particles (snRNP's) from organisms as evolutionarily divergent as insects and dinoflagellates but have been reported not to recognize snRNP's from yeast. The Sm antigen is thought to bind to a conserved snRNA motif that includes the sequence A(U3-6)G. The hypothesis was tested that yeast also contains functional analogues of Sm snRNA's, but that the Sm binding site in the RNA is more strictly conserved than the Sm antigenic determinant. After microinjection of labeled yeast snRNA's into Xenopus eggs or oocytes, two snRNA's from Saccharomyces cerevisiae become strongly immunoprecipitable with human auto-antibodies known as anti-Sm. These each contain the sequence A(U5-6)G, are essential for viability, and are constituents of the spliceosome. At least six other yeast snRNA's do not become immunoprecipitable and lack this sequence; these non-Sm snRNA's are all dispensable.

Animals↗
PubMed1987

A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation.

Splicing of introns from Saccharomyces cerevisiae pre-mRNA requires the conserved sequence TACTAAC; the 3'-most A residue is utilized as the site of branch formation. We showed previously that the transcript from an actin-HIS4 gene fusion containing the mutation TACTAAC to TACTACC (designated C259) is spliced inefficiently, thereby preventing growth on the histidine precursor histidinol. By selecting for growth on histidinol, we have identified a mutant in which the splicing of the C259 transcript is increased fourfold; splicing of other mutated introns is not significantly improved. The mutant locus encodes a trans-acting suppressor. A single mutation, rna16-1, is sufficient for suppression; however, suppression is maximized in heterozygous diploids containing both rna16-1 and the wild-type allele RNA16. In addition, wild-type pre-mRNAs (and lariat intermediates) accumulate in rna16-1 cells. We propose that the RNA16 locus encodes a component of the splicing machinery.

Genes, Fungal↗
PubMed1986

Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome.

Yeast introns contain three highly conserved sequences which are known to be required for splicing of pre-mRNA. Using in vitro mutagenesis, we have synthesized seven point mutations at five different sites in these signals in the yeast actin intron. The mutant introns were then inserted into each of three constructs, which allowed us to assess the consequences both in vivo and in vitro. In virtually every case, we found the efficiency of splicing to be significantly depressed; mature mRNA levels in vivo ranged from 0 to 47% of wild-type. Surprisingly, the tightest mutations were not necessarily at the sites of nucleolytic cleavage and branch formation; these nucleotides are thus highly preferred, but are not absolutely necessary. Moreover, while particular nucleotides are specifically required for the final step in splicing, i.e. 3' cleavage and exon ligation, the predominant consequence of mutation within the conserved signals appears to be the inhibition of assembly of the splicing complex.

Actins↗
PubMed1986

Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity.

Previous work showed that the simple eukaryote Saccharomyces cerevisiae contains a group of RNAs with the general structural properties predicted for small nuclear RNAs (snRNAs), including possession of the characteristic trimethylguanosine 5'-terminal cap. It was also demonstrated that, unlike their metazoan counterparts, the yeast snRNAs are present in low abundance (200-500 molecules per haploid cell). We have now used antibody directed against the 5' cap to investigate the total set size of snRNAs in this organism. We present evidence that the number of distinct yeast snRNAs is on the order of several dozen, that the length of the capped RNAs can exceed 1000 nucleotides, and that the relative abundance of a subset of these RNAs is 1/5th to 1/20th that of the class of snRNAs described previously. These findings suggest that the six highly abundant species of snRNAs (U1-U6) typically reported in metazoans may represent a serious underestimation of the total diversity of snRNAs in eukaryotes.

Cloning, Molecular↗
PubMed1986

Activation of a cryptic TACTAAC box in the Saccharomyces cerevisiae actin intron.

We constructed a translational fusion between the Saccharomyces cerevisiae actin gene and the Escherichia coli beta-galactosidase structural gene such that expression of beta-galactosidase activity required accurate splicing of the actin intron. Using this chimeric gene, we generated a series of internal deletions which removed the TACTAAC box or, in addition, TACTAAC-like sequences within the intron. Analysis of the fusion transcripts produced in these deletions allowed us to conclude that the TACTAAC-like sequence TACTAAG can substitute, albeit inefficiently, for the authentic TACTAAC box in the splicing process. These results indicate that the yeast splicing machinery can utilize a cryptic TACTAAC box, but there are requirements for primary sequence and proper position.

Actins↗
PubMed1985

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.

Base Sequence↗
PubMed1985

A point mutation in the conserved hexanucleotide at a yeast 5' splice junction uncouples recognition, cleavage, and ligation.

We have constructed an actin-HIS4 gene fusion, such that expression of HIS4 requires proper splicing of the actin intron. Using this chimeric gene in an in vivo screen for splicing mutations, we have isolated a G to A transition in the fifth position of the yeast 5' consensus sequence/GTAPyGT. This mutation still allows the junction to be recognized by the splicing machinery, albeit inefficiently. Surprisingly, the fidelity of the 5' endonucleolytic cleavage is also reduced. This results in an incorrect cleavage 6 nucleotides 5' of the 5' junction, at the dinucleotide/AT. Cleavage at this abnormal site does not lead to the production of mature mRNA, although this species appears to be in a lariat structure. The behavior of this mutant argues that recognition of the 5' junction and subsequent cleavage are separable events and, furthermore, that requirements for 3' endonucleolytic cleavage may be more complex than previously imagined.

Actins↗
PubMed1984

Structure of intron-containing tRNA precursors. Analysis of solution conformation using chemical and enzymatic probes.

Using chemical and enzymatic structure-specific probes adapted to rapid gel sequencing techniques, we have analyzed the solution conformations of precursors to two yeast tRNAs which contain an intervening sequence, pre-tRNAPhe and pre-tRNATyr. Interpretation of the data was greatly facilitated by performing direct mature/precursor tRNA comparisons. In addition, the effects of tertiary interactions on probe specificity could be evaluated from the results obtained with mature tRNAPhe, whose crystal structure is known. We find: 1) the folding of the precursor CCA terminus, acceptor stem, T psi C stem, variable loop, anticodon stem, and D stem identical with that of the equivalent regions in the cognate, mature tRNA. 2) The T psi C loop and D loop appear to vary slightly in tertiary structure between mature and precursor species. 3) The precursors contain a helix involving the anticodon triplet and a complementary sequence in the intron. 4) The stability of this helix is much greater for pre-tRNAPhe than for pre-tRNATyr. 5) The splice sites for both precursors are located in single-stranded loops. These results bear out predictions based on genetic analyses and are consistent with the view that recognition of universally conserved features of tRNA structure allows all tRNA precursors containing intervening sequences to be processed by a single splicing apparatus.

Aldehydes↗
PubMed1983

Yeast contains small nuclear RNAs encoded by single copy genes.

We have identified a group of RNA molecules in Saccharomyces cerevisiae that appears to be equivalent to the U class of small nuclear RNAs previously described in other eucaryotes, resembling them in size, metabolic stability, 5' cap structure, presence of modified bases, and nuclear localization. However, the yeast snRNAs differ from their counterparts in several potentially important ways. First, they are present in very low abundance, less than 200 copies per cell, as compared to 10(5)-10(6) for mammalian U1-U6. Second, there appear to be more species in yeast than in any cell type previously examined. Finally, we have cloned five yeast snRNA genes, and find that each is present in a single copy per haploid genome, whereas all previously characterized snRNAs are encoded by multiple (5 to 100) gene copies. The presence of single copy genes in yeast will greatly facilitate the genetic analysis of snRNA function.

Base Sequence↗
PubMed1983

A U4-like small nuclear RNA is dispensable in yeast.

We have cloned a single copy gene that encodes a small nuclear RNA, designated snR3, from the yeast Saccharomyces cerevisiae. This RNA is highly conserved among fungi, and sequence and secondary structure analyses suggest that snR3 is analogous to mammalian U4 snRNA. To determine whether snR3 has an essential function in yeast, the gene (designated SNR3), was disrupted by replacing 35 nucleotides of coding sequences with 2.2 kb of yeast DNA containing the LEU2 gene. Since cells entirely lacking snR3 were expected to be inviable, the nonfunctional gene was used to replace one chromosomal copy in a diploid cell, and the diploid transformants were sporulated. Surprisingly, virtually all tetrads gave rise to four viable spores. Moreover, these haploid strains, which have been shown by DNA blot hybridization to lack an intact copy of the SNR3 gene, and which contain no detectable snR3 transcripts, are indistinguishable from their SNR3+ sister spores under a variety of growth conditions.

Alleles↗
PubMed1981

Yeast ochre suppressor SUQ5-ol is an altered tRNA Ser UCA.

Ochre suppressor tRNA was partially purified from strains of Saccharomyces cerevisiae containing the serine-inserting class III suppressor SUQ5-ol. RNA sequence analysis of this tRNA indicated that the suppressor is derived from a UCA-decoding tRNA Ser by a G leads to U substitution in the middle position of the anticodon. The suppressor further differs from the wild-type UCA-decoding tRNA Ser in that the mutant anticodon lacks the modified uridine found in the wobble position of the wild-type tRNA and contains instead another modification in or near the anticodon.

Anticodon↗
PubMed1981

Yeast tRNA precursor mutated at a splice junction is correctly processed in vivo.

Yeast mutants with decreased expression of a tRNATyr gene were obtained by selection for functional inactivation of the tyrosine-inserting ochre suppressor SUP4 and subsequent screening for production of the tRNA gene product in vivo. One mutant with reduced suppressor activity was characterized by a decreased quantity of the suppressor-specific tRNA; a precursor to this tRNA, matured at both 5' and 3' termini but still containing a 14-nucleotide intervening sequence, was present in an amount greater than 7-fold that in the parent. By RNA sequence analysis of the accumulated precursor, we have identified the mutation as an A leads to G transition at the 5' splice junction. Similar analysis of the mature tRNA produced in this mutant demonstrated that the intervening sequence was accurately excised. We conclude that the specific sequence of nucleotides at this splice junction affects the efficiency but not the fidelity of processing.

Base Sequence↗