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C Guthrie

Publications and source records attributed to C Guthrie.

At least 73 records · Page 4Linked to original sources
PubMed1991

Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs.

U4 and U6 small nuclear RNAs are associated by an extensive base-pairing interaction that must be disrupted and reformed with each round of splicing. U4 mutations within the U4/U6 interaction domain destabilize the complex in vitro and cause a cold-sensitive phenotype in vivo. Restabilization of the U4/U6 helix by dominant (gain-of-function), compensatory mutations in U6 results in wild-type growth. Cold-insensitive growth can also be restored by two classes of recessive (loss-of-function) suppressors: (1) mutations in PRP24, which we show to be a U6-specific binding protein of the RNP-consensus family; and (2) mutations in U6, which lie outside the interaction domain and identify putative PRP24-binding sites. Destabilization of the U4/U6 helix causes the accumulation of a PRP24/U4/U6 complex, which is undetectable in wild-type cells. The loss-of-function suppressor mutations inhibit the binding of PRP24 to U6, and thus presumably promote the release of PRP24 from the PRP24/U4/U6 complex and the reformation of the base-paired U4/U6 snRNP. We propose that the PRP24/U4/U6 complex is normally a highly transient intermediate in the spliceosome cycle and that PRP24 promotes the reannealing of U6 with U4.

Amino Acid Sequence↗
PubMed1990

Human U2 snRNA can function in pre-mRNA splicing in yeast.

The removal of introns from messenger RNA precursors requires five small nuclear RNAs (snRNAs), contained within ribonucleoprotein particles (snRNPs), which complex with the pre-mRNA and other associated factors to form the spliceosome. In both yeast and mammals, the U2 snRNA base pairs with sequences surrounding the site of lariat formation. Binding of U2 snRNP to the highly degenerate branchpoint sequence in mammalian introns is absolutely dependent on an auxiliary protein, U2AF, which recognizes a polypyrimidine stretch adjacent to the 3' splice site. The absence of this sequence motif in yeast introns has strengthened arguments that the two systems are fundamentally different. Deletion analyses of the yeast U2 gene have confirmed that the highly conserved 5' domain is essential, although the adjacent approximately 950 nucleotides can be deleted without any phenotypic consequence. A 3'-terminal domain of approximately 100 nucleotides is also required for wild-type growth rates; the highly conserved terminal loop within this domain (loop IV) may provide specific binding contacts for two U2-specific snRNP proteins. We have replaced the single copy yeast U2 (yU2) gene with human U2 (hU2), expecting that weak or no complementation would provide an assay for cloning additional splicing factors, such as U2AF. We report here that hU2 can complement the yeast deletion with surprising efficiency. The interactions governing spliceosome assembly and intron recognition are thus more conserved than previously suspected. Paradoxically, the conserved loop IV sequence is dispensable in yeast.

Base Sequence↗
PubMed1990

A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing.

We previously described a dominant suppressor of the splicing defect conferred by an A----C intron branchpoint mutation in S. cerevisiae. Suppression occurs by increasing the frequency with which the mutant branchpoint is utilized. We have now cloned the genomic region encoding the prp16-1 suppressor function and have demonstrated that PRP16 is essential for viability. A 1071 amino acid open reading frame contains sequence motifs characteristic of an NTP binding fold and further similarities to a superfamily of proteins that includes members with demonstrated RNA-dependent ATPase activity. A single nucleotide change necessary to confer the prp16-1 suppressor phenotype results in a Tyr----Asp substitution near the "A site" consensus for NTP binding proteins. We propose that PRP16 is an excellent candidate for mediating one of the many ATP-requiring steps of spliceosome assembly and that accuracy of branchpoint recognition may be coupled to ATP binding and/or hydrolysis.

Adenosine Triphosphatases↗
PubMed1990

Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site.

Human autoantibodies of the Sm specificity recognize a conserved set of proteins found in the U class small nuclear ribonucleoproteins (U snRNPs), key trans-acting factors involved in the splicing of mRNA precursors. The Sm protein binding site in U snRNAs is unusual because of its single-stranded nature and its simple sequence motif (AU5-6GPu). Here we use genetics to probe this specific protein-RNA interaction by saturation mutagenesis of the Sm binding site of the Saccharomyces cerevisiae U5 snRNA. The assay system used to analyze these mutations takes advantage of a conditionally expressed U5 gene which does not support growth under non-permissive conditions; U5 genes containing Sm site mutations were tested for their ability to complement this lethal phenotype. Our results indicate that the Sm binding site is remarkably tolerant to mutation despite its high degree of conservation, suggesting that relatively few or redundant specific contacts can determine recognition of single-stranded RNA by protein. A complementary biochemical analysis of these mutants demonstrates that integrity of the Sm site is necessary for snRNP stability in vivo and in vitro.

Autoantigens↗
PubMed1990

Intraluminal vascular ultrasound: preliminary report of dimensional and morphologic accuracy.

The role of intraluminal ultrasound for diagnosis and monitoring treatment of vascular disease has yet to be defined. This study evaluated the dimensional precision and morphologic accuracy of an intraluminal ultrasound system which consists of a 5.5 French external diameter ultrasonic catheter with a central lumen for passage of a guidewire. Ultrasound images from five in-vitro human and three porcine arterial segments and two in-vivo arteriosclerotic canine arteries were compared to dimensions obtained from arteriograms and from sections of the specimens. Each gross and histological specimen and ultrasound image was scaled, photographed and enlarged up to 20 times and measured for vessel intraluminal and adventitial or outer diameter and wall thickness. Intraluminal and outer diameters and wall thickness from normal in-vitro specimens correlated significantly with dimensions obtained from histologic specimens (r = 0.99, p less than 0.005 for internal and outer diameters and r = 0.73, p less than 0.005 for wall thickness). The mean differences of luminal diameters measured from the vessels supported within a silicone rubber mold was 0.05 +/- 0.09 mm (n = 20). Diameters of outer diameter and wall thickness were less reliably defined, the average margin of error being 0.49 +/- 0.39 mm and 0.29 +/- 0.26 mm, respectively. The mean difference between in-vivo ultrasound and arteriographic diameters was 0.61 +/- 0.38 mm (n = 12). Correlation of luminal diameters between ultrasound and arteriogram was significant (r = 0.76, p less than 0.02). The ultrasound images also differentiated a laminated appearance of normal vessel anatomy from non-uniform or dense signals seen in atherosclerotic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiography↗
PubMed1990

Multiple roles for U6 snRNA in the splicing pathway.

U6 is the most highly conserved of the five spliceosomal RNAs. It is associated with U4 by an extensive base-pairing interaction, which is disrupted immediately prior to the first nucleolytic step of splicing. It has been proposed that this event activates catalysis by unmasking U6. Using a combination of doped synthesis and site-directed mutagenesis to generate point mutations in U6, we have now identified 12 positions, in three domains, at which single nucleotide substitutions or deletions result in lethal or temperature-sensitive phenotypes. Biochemical analysis demonstrates that most of these mutants retain the ability to assemble into U4/U6 and U4/U5/U6 snRNPs. Notably, although mutations at three positions in U6 that base-pair with U4 are lethal, mutations in the complementary residues in U4 are fully viable. Furthermore, compensatory mutations in U4 that restore base-pairing fail to suppress the phenotypes of the U6 mutations. This demonstrates a function for U6 independent of its role in base-pairing. Remarkably, two of the three essential regions in U6 identified genetically correspond to intron insertion points in two yeast species. A temperature-sensitive mutation at one of these sites is defective in the second step of splicing in vitro.

Base Sequence↗
PubMed1990

Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.

U4 small nuclear RNA (snRNA) contains two intramolecular stem-loop structures, located near each end of the molecule. The 5' stem-loop is highly conserved in structure and separates two regions of U4 snRNA that base-pair with U6 snRNA in the U4/U6 small nuclear ribonucleoprotein particle (snRNP). The 3' stem-loop is highly divergent in structure among species and lies immediately upstream of the binding site for Sm proteins. To investigate the function of these two domains, mutants were constructed that delete the yeast U4 snRNA 5' stem-loop and that replace the yeast 3' stem-loop with that from trypanosome U4 snRNA. Both mutants fail to complement a null allele of the yeast U4 gene. The defects of the mutants have been examined in heterozygous strains by native gel electrophoresis, glycerol gradient centrifugation, and immunoprecipitation. The chimeric yeast-trypanosome RNA does not associate efficiently with U6 snRNA, suggesting that the 3' stem-loop of yeast U4 snRNA might be a binding site for a putative protein that facilitates assembly of the U4/U6 complex. In contrast, the 5' hairpin deletion mutant associates efficiently with U6 snRNA. However, it does not bind the U4/U6-specific protein PRP4 and does not assemble into a U4/U5/U6 snRNA. Thus, we propose that the role of the PRP4 protein is to promote interactions between the U4/U6 snRNP and the U5 snRNP.

Base Sequence↗
PubMed1990

Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position.

Vertebrate genes coding for U6 small nuclear RNA are transcribed by RNA polymerase III (pol III), using only upstream promoter elements rather than the A and B block internal control regions typical of most pol III transcription units. We show that expression of the U6 gene from the yeast Saccharomyces cerevisiae has two unexpected features: it requires a B block promoter element, and this element is located in a novel position, 120 bp downstream of the coding region. In tRNA genes, the B block is the primary binding site for transcription factor (TF) IIIC, whose function is to promote the subsequent binding of TFIIIB. Both factors are thus implicated in yeast U6 gene transcription. We present a model of the U6 transcription complex based on the structure of yeast and vertebrate U6 promoters.

Base Sequence↗
PubMed1990

Intravascular ultrasound: a new potential modality for angioplasty guidance.

Current angioplasty devices are limited by significant rates of arterial perforation and dissection, due to inadequate techniques of guidance, and by restenosis, which may be partly attributed to inadequate debulking of lesions. This paper describes the authors' initial experience in-vitro and in-vivo with intravascular ultrasound as a possible method of enhancing the three-dimensional guidance of devices through atherosclerotic obstructions. Using an in-vitro model they correlated the dimensions and histologic morphology of animal and human arteries with ultrasound images of the specimens. Additional in-vivo evaluations of this technology in canine arteriosclerotic and human atherosclerotic arteries preliminarily support the hypothesis that intravascular ultrasound defines the transmural arterial morphology and may enhance the accuracy of angioplasty procedures. Simultaneous imaging with angioscopy and intravascular ultrasound is demonstrated as a potential method of accurately defining both intraluminal and transmural arterial wall characteristics.

Angioplasty, Balloon↗
PubMed1989

Small nuclear RNAs from budding yeasts: phylogenetic comparisons reveal extensive size variation.

Homologues of each of the five metazoan snRNAs required for pre-mRNA splicing have recently been identified in the budding yeast Saccharomyces cerevisiae on the basis of shared structural elements and evidence of similar roles during splicing. However, the spliceosomal snRNAs in this yeast are up to six times larger than their mammalian counterparts, suggesting that they may perform additional, perhaps species-specific, functions in the pre-mRNA processing pathway. We have undertaken a survey of 23 other budding yeasts to determine whether increased snRNA size is unique to Sacch. cerevisiae and, if not, to look for common structural motifs among homologous snRNAs. Our studies reveal that the spliceosomal snRNAs exhibit a surprising degree of size variation among these species. Furthermore, partial sequence analysis has identified a specific domain in the U6 snRNA which accounts for the observed size polymorphisms.

Base Sequence↗
PubMed1988

Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides.

Yeast U2 snRNA (1175 nucleotides) is six times larger than its mammalian counterpart (188 nucleotides). Using deletion analysis, we show that the molecule can be divided into three phenotypically distinct domains. As expected, the highly conserved 5' domain (approximately 120 nucleotides) is absolutely essential for viability. Surprisingly, however, deletion of the central 945 nucleotides has no effect on growth rate. In contrast, removal of sequences in the 3' terminal 110 nucleotides results in low numbers of slow-growing colonies; these cells contain U2 with altered 3' ends. This domain can be folded into a secondary structure that strongly resembles the 3' terminal stem-loop IV of human U2. We conclude that yeast U2 contains two functionally important elements. While the 5' domain is known to be directly involved in the splicing reaction, the 3' domain may function primarily in the generation of stable small nuclear ribonucleoprotein particles.

Alleles↗
PubMed1988

Spliceosomal RNA U6 is remarkably conserved from yeast to mammals.

The small nuclear RNA U6 and its gene have been isolated from yeast. In striking contrast to other yeast spliceosomal RNAs, U6 is very similar in size, sequence and structure to its mammalian homologue. The single-copy gene is essential. These properties suggest a central role in pre-mRNA processing. An extensive base-pairing interaction with U4 snRNA is described; the destabilization of the U4/U6 complex seen during splicing thus requires a large conformational change.

Base Sequence↗
PubMed1988

5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements.

Using a strategy of compensatory nucleotide changes between yeast U1 and a 5' splice site, we have analyzed the contribution of base-pairing to the efficiency and fidelity of pre-mRNA splicing in vivo. Watson-Crick base-pairing interactions with U1 can be demonstrated at intron positions 1 and 5 but not at position 4. Moreover, restoration of the ability to pair with U1 is not sufficient to restore activity in the second step of splicing to intron position 1 mutants. Finally, in contrast to recent observations in mammalian systems, we find that the precise position of 5' splice site cleavage is not determined solely by the base-pairing interaction with U1. Rather, the presence of a G residue at position 5 is required for the correct localization of the nucleolytic event. Taken together, these results indicate that the demands for 5' splice site selection and utilization are more complex than a simple maximization of Watson-Crick interactions with U1.

Base Composition↗
PubMed1988

Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant.

Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature. None of these RNAs associates with the Sm antigen. To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs. Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site. By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable. We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles. A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10. This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs.

Base Sequence↗