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C Guthrie

Publications and source records attributed to C Guthrie.

At least 37 records · Page 2Linked to original sources
PubMed1997

Alternative 3'-end processing of U5 snRNA by RNase III.

The cellular components required to form the 3' ends of small nuclear RNAs are unknown. U5 snRNA from Saccharomyces cerevisiae is found in two forms that differ in length at their 3' ends (U5L and U5S). When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5. The two forms of U5 are produced in vitro by alternative 3'-end processing. A temperature-sensitive mutation in the RNT1 gene encoding RNase III blocks accumulation of U5L in vivo. In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III. These results identify RNase III as a trans-acting factor involved in 3'-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.

Base Sequence↗
PubMed1997

Exportin 1 (Crm1p) is an essential nuclear export factor.

Nuclear protein export is mediated by nuclear export signals (NESs), but the mechanisms governing this transport process are not well understood. Using a novel protein export assay in S. cerevisiae, we identify CRM1 as an essential mediator of nuclear protein export in yeast. Crm1p shows homology to importin beta-like transport factors and is able to specifically interact with both the NES motif and the Ran GTPase. A mutation in the shuttling protein Crm1p affects not only protein export, but also mRNA export, indicating that these pathways are tightly coupled in S. cerevisiae. The presented data are consistent with the conclusion that Crm1p is a carrier for the NES-mediated protein export pathway. We propose CRM1 be renamed exportin 1 (XPO1).

Biological Transport↗
PubMed1996

Essential yeast protein with unexpected similarity to subunits of mammalian cleavage and polyadenylation specificity factor (CPSF).

The 3' ends of most eukaryotic messenger RNAs are generated by internal cleavage and polyadenylation. In mammals, there is a strict dependence of both reactions on the sequence AAUAAA, which occurs upstream of polyadenylation [poly(A)] sites and which is recognized by CPSF. In contrast, cis-acting signals for yeast 3'-end generation are highly divergent from those of mammals, suggesting that trans-acting factors other than poly(A) polymerase would not be conserved. The essential yeast protein Brr5/Ysh1 shows sequence similarity to subunits of mammalian CPSF and is required for 3'-end processing in vivo and in vitro. These results demonstrate a structural and functional conservation of the yeast and mammalian 3'-end processing machineries despite a lack of conservation of the cis sequences.

Amino Acid Sequence↗
PubMed1996

The essential yeast RNA binding protein Np13p is methylated.

Arginine methylation is a prevalent modification found in many RNA binding proteins, yet little is known about its functional consequences. Using a monoclonal antibody, 1E4, we have shown that the yeast NPL3 gene product Np13p, an essential RNA binding protein with repeated RGG motifs, is arginine-methylated in vivo. The 1E4 epitope can be generated by incubating recombinant Np13p with partially purified bovine arginine methyltransferase block this reaction. Np13p methylation requires S-adenosyl-L-methionine and also occurs in yeast extracts. An Np13p deletion mutant lacking the RGG domain is not a substrate for methylation, suggesting that the methylation sites lie within the RGG motifs. The discovery of arginine methylation in a genetically tractable organism provides a powerful entrée to understanding the function of this modification, particularly in view of the many roles postulated for Np13p in RNA processing and transport. The recent discovery of phosphorylated serine residues within the RGG domain suggests a hypothesis in which a molecular switch governed by methylation and phosphorylation regulates the biochemical properties of the Np13p RGG domain.

Amino Acid Sequence↗
PubMed1996

Transcriptional pulse-chase analysis reveals a role for a novel snRNP-associated protein in the manufacture of spliceosomal snRNPs.

Vertebrate spliceosomal snRNAs associate with a conserved set of proteins, the Sm proteins, via a conserved RNA sequence, the Sm site. Assembly of this complex is required for the accumulation of stable snRNPs, hypermethylation of the 5' cap structure and nuclear import of the resultant particles. The function of individual core snRNP proteins is poorly understood, in part because of the difficulty of selectively inactivating individual polypeptides in vivo. Using a transcriptional pulse-chase method we have defined for the first time the steps of snRNP biogenesis in Saccharomyces cerevisiae. We describe a novel component of spliceosomal snRNPs, Brr1, which is distinct in sequence from Sm core proteins and yet which shares many of their properties, as well as a genetic interaction with the yeast homolog of Sm D1 core protein. Through a kinetic analysis of snRNP formation in wild-type and brr1 mutant cells we demonstrate specific defects in a subset of steps in the brr1 mutant: newly synthesized snRNAs are destabilized and 3'-end processing is slowed, whereas the cap hypermethylation reaction is unaffected. Notably, the stability of mature particles, as measured by promoter shut-off experiments, is normal in the absence of the Brr1 snRNP protein.

Amino Acid Sequence↗
PubMed1996

Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations.

Genetic approaches in Saccharomyces cerevisiae have identified 38 genes required for efficient RNA splicing. The majority have been found by screening (high) temperature-sensitive (ts) mutants for those defective in splicing, an approach limited by the presence of ts hotspots and by the fact that many essential genes rarely mutate to the ts phenotype. To identify novel genes, we screened a collection of 340 cold-sensitive (cs) mutants for those that exhibited diminished splicing of several pre-mRNAs. We isolated 12 mutants in nine complementation groups. Four of these affected known genes (PRP8, PRP16, PRP22, PRP28), three of which encode RNA helicase homologues. Five genes are novel (BRR1, BRR2, BRR3, BRR4, BRR5; Bad Response to Refrigeration); mutations in these genes inhibited splicing before the first chemical step of the reaction. Analysis of BRR2 revealed it to encode an essential member of a new class of RNA helicase-like proteins that includes the yeast antiviral protein Ski2. These data validate the use of cs mutants in genetic screens and raise the possibility that RNA helicase family members are particularly prone to mutation to cold sensitivity.

Amino Acid Sequence↗
PubMed1996

Mutagenesis of the yeast gene PRP8 reveals domains governing the specificity and fidelity of 3' splice site selection.

PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the uridine tract that precedes most yeast 3' slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3' splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3' splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the prp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3' splice site selection and fidelity of 3' splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing.

Amino Acid Sequence↗
PubMed1995

Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast.

Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prp17-1, and prp18-1) are known to confer a specific block to the second chemical step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerevisiae. Previous studies showed that Prp16p and Prp18p are required solely for the second step in vitro. The RNA-dependent ATPase, Prp16p, functions at a stage in splicing when ATP is required, whereas Prp18p functions at an ATP-independent stage. Here we use immunodepletion to show that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require both Prp17p and ATP for slicing complementation, whereas extracts depleted of Slu7p require only the addition of Slu7p. These different ATP requirements suggest that Prp16p and Prp17p function before Prp18p and Slu7p. Although SLU7 encodes an essential gene product, we find that a null allele of prp17 is temperature-sensitive for growth and has a partial splicing defect in vitro. Finally, high-copy suppression experiments indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that these four factors may function within a multi-component complex that has both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.

Adenosine Triphosphate↗
PubMed1995

A novel role for a U5 snRNP protein in 3' splice site selection.

The choice of a 3' splice site in Saccharomyces cerevisiae introns involves recognition of a uridine-rich tract upstream of the AG dinucleotide splice junction. By isolating mutants that eliminate the normal preference for uridine-containing 3' splice sites in a cis-competition, we identified a mutation that is an allele of PRP8, prp8-101. This was unexpected because previous analysis has demonstrated that the U5 snRNP protein encoded by PRP8 is required for spliceosome assembly prior to the first catalytic step of splicing. In contrast, the uridine recognition defect caused by the prp8-101 mutation selectively inhibits the second catalytic step of splicing. This defect is seen not only in 3' splice site cis-competitions but also in the splicing of an unusual intron in the TUB3 gene and in the ACT1 intron when utilization of its 3' splice site is rate limiting for splicing. Consistent with a direct role in 3' splice site selection, Prp8 can be cross-linked to the 3' splice site during the splicing reaction. These data demonstrate a novel function for Prp8 in 3' splice site recognition and utilization.

Actins↗
PubMed1995

Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs.

A mutation (U4-G14C) that destabilizes the base-pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs. An analysis of suppressors of this cold-sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6. It was proposed that Prp24 must be released to form a fully base-paired U4/U6 snRNP. By using a chemical probing method we have tested the prediction that nucleotides A40-C43 in U6 mediate the binding of Prp24. Consistent with the location of recessive suppressors in U6, we find that residues A40-C43 are protected from chemical modification in U4/U6 complexes from the U4-G14C mutant but not from the wild-type or suppressor strains carrying mutations in U6 or PRP24. Furthermore, we find that base-pairing is substantially disrupted in the mutant complexes. Notably, the base-paired structure is restored in recessive suppressors despite the presence of a mismatched base-pair at the U4-G14C site. Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.

Base Sequence↗
PubMed1995

Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing.

For the second catalytic step of pre-mRNA splicing to occur, a 3' splice site must be selected and juxtaposed with the 5' exon. Four proteins, Prp16p, Slu7p, Prp17p, Prp18p, and an integral spliceosomal protein, Prp8p, are known to be required for the second catalytic step. prp8-101, an allele of PRP8 defective in 3' splice site recognition, exhibits specific genetic interactions with mutant alleles of the other second step splicing factors. The prp8-101 mutation also results in decreased crosslinking of Prp8p to the 3' splice site. To determine the role of the step-two-specific proteins in 3' splice site recognition and in binding of Prp8p to the 3' splice site, we performed crosslinking studies in mutant and immunodepleted extracts. Our results suggest an ordered pathway in which, after the first catalytic step, Prp16p crosslinks strongly to the 3' splice site and Prp8p and Slu7p crosslink weakly. ATP hydrolysis by Prp16p affects a conformational change that reduces the crosslinking of Prp16p with the 3' splice site and allows stronger crosslinking of Prp8p and Slu7p. Thus, the 3' splice site appears to be recognized in two stages during the second step of splicing. Strong 3' splice site crosslinking of Prp8p and Slu7p also requires the functions of Prp17p and Prp18p. Therefore, Prp8p and Slu7p interact with the 3' splice site at the latest stage of splicing prior to the second catalytic step that can currently be defined, and may be at the active site.

Adenosine Triphosphatases↗
PubMed1994

PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro.

We previously reported the isolation of PRP28, a gene in Saccharomyces cerevisiae whose activity is required for the first step of nuclear mRNA splicing in vivo. Sequence analysis revealed that PRP28 is included in the 'DEAD-box' gene family, members of which are thought to function as ATP-dependent RNA helicases. Genetic interactions led us to suggest that PRP28 is functionally associated with the U4/U5/U6 snRNP. We have now purified the PRP28 protein from S. cerevisiae and demonstrated that it is required for the first step of splicing in vitro. Interestingly, PRP28 is not a stably associated snRNP protein. Strand displacement assays indicate that PRP28 does not exhibit RNA helicase activity, suggesting that an additional factor or factors may be required for its activation.

Adenosine Triphosphatases↗
PubMed1994

Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome.

Putative components of the spliceosomal active site include a bulged helix between U2 and U6 snRNAs (U2-U6 helix I) and the adjacent ACAGAG hexanucleotide in U6. We have developed an in vivo, bimolecular randomization-selection method to functionally dissect these elements. Although a portion of U2-U6 helix I resembles the G-binding site of group I introns, the data are inconsistent with an analogous functional role for this structure in the spliceosome. Instead, analysis of several novel covariants supports the existence of a structure in which the helix I bulge engages in a tertiary interaction with the terminal residue of the U6 hexanucleotide (ACAGAG). Such a higher order structure, together with other known interactions, would juxtapose the two clusters of residues of the U2-U6 complex that are specifically required for the second chemical step of pre-mRNA splicing with the 3' splice site. Indeed, mutations in the residues that participate in the tertiary interaction affect both the efficiency and fidelity of 3' splice site usage.

Alleles↗
PubMed1994

Genetic interactions between the yeast RNA helicase homolog Prp16 and spliceosomal snRNAs identify candidate ligands for the Prp16 RNA-dependent ATPase.

Pre-mRNA splicing occurs in a large and dynamic ribonucleoprotein complex, the spliceosome. Several protein factors involved in splicing are homologous to a family of RNA-dependent ATPases, the so-called DEAD/DEAH proteins. A subset of these factors exhibit RNA helicase activity in vitro. The DEAD/DEAH proteins involved in splicing are thought to mediate RNA conformational rearrangements during spliceosome assembly. However, the RNA ligands for these factors are currently unknown. Here, we present genetic evidence in Saccharomyces cerevisiae for a functional interaction between the DEAH protein Prp16, and the U6 and U2 spliceosomal snRNAs. Using a library of mutagenized U6 snRNA genes, we have identified 14 strong suppressors of the cold-sensitive (cs) allele, prp16-302. Remarkably, each suppressor contains a single nucleotide deletion of 1 of the 6 residues that lie immediately upstream of a sequence in U6 that interacts with the 5' splice site. Analysis of site-directed mutations revealed that nucleotide substitutions in the adjacent U2-U6 helix I structure also suppress prp16-302, albeit more weakly. The U6 suppressors tested also partially reverse the phenotype of two other cs alleles, prp16-1 and prp16-301, but not the four temperature-sensitive alleles tested. Finally, overexpression of each cs allele exacerbates its recessive growth phenotype and confers a dominant negative cs phenotype. We propose that the snRNA suppressors function by destabilizing an interaction between the U2-U6 complex and a hypothetical factor (X), which is trapped by cs mutants of PRP16. The phenotypes of overexpressed prp16 alleles are consistent with the model that this trapped interaction inhibits the dissociation of Prp16 from the spliceosome. We discuss the intriguing possibility that factor X is Prp16 itself.

Adenosine Triphosphatases↗
PubMed1994

Architecture of the U5 small nuclear RNA.

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

Autoantigens↗
PubMed1994

CRP levels as a measure of surgical trauma: a comparison of different general surgical procedures.

Acute phase proteins are released into the circulation as part of the metabolic response to trauma. C reactive protein (CRP) has been shown to be the most specific and sensitive indicator of trauma. We measured pre- and postoperative CRP levels in patients undergoing varicose vein surgery, inguinal herniorrhaphy, laparoscopic cholecystectomy and open cholecystectomy. A significant difference is shown between the levels found in those undergoing varicose vein, hernia surgery or open cholecystectomy; however, there is no significant difference in the CRP levels between open and laparoscopic cholecystectomy.

C-Reactive Protein↗