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C Guthrie

Publications and source records attributed to C Guthrie.

At least 19 recordsLinked to original sources
PubMed1992

A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome.

Prior to the chemical steps of mRNA splicing, the extensive base-pairing interaction between the U4 and U6 spliceosomal snRNAs is disrupted. Here, we use a mutational analysis in yeast to demonstrate a conserved base-pairing interaction between the U6 and U2 snRNAs that is mutually exclusive with the U4-U6 interaction. In this novel pairing, conserved sequences in U6 interact with a sequence in U2 that is immediately upstream of the branch point recognition region. Remarkably, the residues in U6 that can be consequently juxtaposed with the intron substrate include those that have been proposed previously to be catalytic. Both the first and second steps of splicing are inhibited when this base-paired structure is mutated. These observations, together with the high conservation of the U2-U6 structure, lead us to propose that it might be a component of the spliceosomal active site.

Base Sequence↗
PubMed1992

Human and human-yeast chimeric U6 snRNA genes identify structural elements required for expression in yeast.

U6 is the most highly conserved spliceosomal snRNA. Previous mutational studies have shown that the majority of essential residues in U6 are located in a region of 35 nucleotides encompassing a conserved hexanucleotide and stem I and stem II of the U4-interaction domain. Although the yeast and human U6 RNAs are 80% identical in this region, the human U6 gene cannot functionally replace the yeast gene in vivo. The human gene is not transcribed when placed in the context of yeast flanking sequences. Transcription of the human gene, but not its function, can be stimulated by the introduction of an A block promoter element in the U6 coding region. Using a set of human-yeast chimeras, we show that the 5' domain and the 3' terminal region of the human U6 gene can each functionally replace the corresponding yeast domains. However, a combination of both domains in a single molecule is lethal. The basis of the inability of the human U6 snRNA to function in yeast cells is discussed.

Base Composition↗
PubMed1992

A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.

Adenosine Triphosphatases↗
PubMed1992

An essential splicing factor, SLU7, mediates 3' splice site choice in yeast.

Recently, we have reported the identification of several genes that exhibit genetic interactions with the U5 snRNA. Two of these genes, SLU4 and SLU7 (SLU: synergistic lethal with U5 snRNA), encode products required for the second catalytic step of splicing. To analyze the specific roles of SLU4 and SLU7, we have determined how mutants influence the relative usage of competing 3' splice sites. We find that mutations in SLU7 eliminate the normal 20-fold preference for 3' splice sites located > 22 nucleotides downstream of the branchpoint. In contrast, mutations in SLU4 inhibit usage of all 3' splice sites, regardless of their location. This suggests that SLU7 is involved in the process of 3' splice site choice, whereas SLU4 fulfills a generic requirement for the second step. We show that SLU7 is an essential gene that contains a small motif with striking similarity to the cysteine-rich zinc knuckle of retroviral nucleocapsid proteins, which has been implicated in RNA binding. Mutational analysis of SLU7 indicates that this motif influences the efficiency, but not the sequence specificity, of 3' splice site selection. The identification of a component of the constitutive splicing machinery that can promote 3' splice site choice has potentially important implications for alternative splicing.

Amino Acid Sequence↗
PubMed1992

Morbidity in patients on L-thyroxine: a comparison of those with a normal TSH to those with a suppressed TSH.

OBJECTIVE: Patients on L-thyroxine with a 'suppressed' TSH (< 0.05 mU/l) were compared to those in whom TSH was detectable but not elevated (0.05-4.0 mU/l), with regard to morbidity data. DESIGN: Biochemical data from Tayside Thyroid Register was matched to hospital admissions data obtained from Health Board Statistics. PATIENTS: The patients were identified from those registered on the computerized Tayside Register. MEASUREMENTS: Serum T4 and TSH assays, clinical assessment scores, and admission records with regard to ischaemic heart disease, overall fractures, fractured neck of femur and breast carcinoma. RESULTS: Over one year, 1180 patients on thyroxine replacement had clinical and biochemical assessment; 59% had a suppressed TSH and 38% 'normal' TSH. Patients with a suppressed TSH exhibited higher median serum thyroxine levels (146 nmol/l, range 77-252 vs 119 nmol/l, 58-224; P < 0.001). Patients under the age of 65 years on L-thyroxine had an increased risk of ischaemic heart disease compared to the general population (female 2.7 vs 0.7%, P < 0.001; male 6.4 vs 1.7%, P < 0.01), but the risk was no different between those with suppressed and normal TSH. There was no increase in risk for overall fracture, fractured neck of femur or breast carcinoma in those on thyroxine with suppressed or normal TSH. CONCLUSION: Patients under the age of 65 years on L-thyroxine had an increased risk of ischaemic heart disease. There was no excess of fractures in patients on L-thyroxine even if the TSH is suppressed.

Aged↗
PubMed1992

Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing.

To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA. We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA. SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 [U. Vijayraghavan, M. Company, and J. Abelson, Genes Dev. 3:1206-1216, 1989]) are required only for the second step of splicing. Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4. We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing.

Base Sequence↗
PubMed1992

A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome.

PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.

Adenosine Triphosphatases↗
PubMed1991

More than half of yeast U1 snRNA is dispensable for growth.

Yeast U1 snRNA (568 nucleotides) is 3.5-fold larger than its mammalian counterpart (164 nucleotides) and contains apparent sequence homology only at the 5' and 3' ends. We have used deletion analysis to determine whether the yeast-specific U1 sequences play essential roles in vivo. Yeast cells carrying a deletion of more than 60% (355 nucleotides) of the single-copy U1 gene are viable, though slow-growing, while a deletion of 316 nucleotides allows essentially wild-type growth. The boundaries of the viable deletions define a dispensable internal domain which comprises sequences unique to yeast. In contrast, the essential 5' and 3' terminal domains correspond to phylogenetically conserved sequences and/or structures previously implicated in RNA:RNA and RNA:protein interactions. The minimal essential sequences of yeast U1 can be drawn in a secondary structure which resembles metazoan U1 in four of seven structural domains.

Base Sequence↗
PubMed1991

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein.

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.

Adenosine Triphosphate↗
PubMed1991

PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome.

The assembly of the spliceosome is an ATP-dependent process. The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family. The protein has now been purified and shown to exhibit RNA-dependent ATPase activity. PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro. This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome. PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.

Adenosine Triphosphatases↗
PubMed1991

A U-rich tract enhances usage of an alternative 3' splice site in yeast.

There has been a long-standing belief that the mechanisms of mammalian and yeast splicing differ fundamentally in their requirement for a pyrimidine-rich motif preceding the 3' splice site. Using an in vivo assay, we have tested the influence of uridine content on competition between alternative 3' splice sites in yeast. We find that a uridine-rich tract preceding a PyAG greatly enhances its ability to compete as a splice acceptor. Moreover, a proximal PyAG is often overlooked if a more distal PyAG occurs in a superior sequence context; this observation cannot be accounted for by simple scanning models. Finally, we show that a distal (greater than 30 nucleotide) 3' splice site that is not preceded by uridines is a poor substrate for the second step of splicing; this argues that recognition of a uridine-rich motif is required for effective identification and utilization of distant splice sites.

Actins↗
PubMed1991

Stenosis following laser thermal angioplasty--a blinded controlled randomized study between aspirin against Probucol.

Long-term patency of vascular angioplasties is limited by stenosis from neointimal hyperplasia or progressive arteriosclerosis. This study evaluated the hypothesis that ASA (an antiprostaglandin) or Probucol (an antioxidant) may be useful in preventing stenosis following laser thermal angioplasty. Aortoiliac arteriosclerosis was induced in 17 female New Zealand white rabbits with a combination of endothelial denudation (4 Fr. Fogarty balloon catheters) and 2% cholesterol-supplemented diet for 6 weeks. All rabbits then underwent arteriography and were immediately randomized to one of three groups: Group I (n = 6), control, laser but no adjunctive therapy; Group II (n = 5), laser and ASA 10 mg/kg/day; Group III (n = 6), laser and 1% Probucol diet. Retrograde laser thermal angioplasty was performed with a 1-mm coronary Laserprobe. Six watts of argon laser energy was delivered for a single 5-sec pulse using a continuous motion from the right common iliac artery to the aorta. Pre- and postlaser arteriography was performed in all groups. Rabbits were sacrificed 1 month following laser angioplasty, and aortoiliac sections were taken after in situ perfusion fixation with paraformaldehyde and 0.05% glutaraldehyde. Serial 5-mm segments, proximal to the aortic bifurcation and distally from the same, were taken, stained, and evaluated by quantitative morphometry. From each segment, lesion area and lesion area/internal elastic laminae area were evaluated and compared. Serum cholesterol increased from 60.8 +/- 19.5 to 1494.7 +/- 12.7 mg% following institution of the cholesterol diet (P less than 0.05). Arteriosclerotic lesions were observed in all rabbits and maximally located around the common iliac artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Angioplasty, Laser↗
PubMed1991

A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family.

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which the first step of mRNA splicing is inhibited. The growth and splicing defects are recessive and cosegregate, thus defining a single essential gene (PRP28). The wild-type PRP28 gene was cloned, and sequence analysis reveals extensive homology to a family of proteins that are thought to function as ATP-dependent RNA helicases. The cold sensitivity is caused by a glycine-to-glutamic acid change in a conserved sequence motif. Interestingly, double mutants containing conditional alleles of PRP28 and PRP24, which encodes a U6 snRNA-binding protein, are inviable. In addition, a suppressor of prp28-1 is a mutant allele of PRP8, which encodes a U5 protein, thus linking PRP28 with U5. These data are consistent with a scenario in which PRP28 acts to unwind the U4/U6 base-pairing interaction in the U4/U6/U5 snRNP, facilitating the first covalent step of splicing.

Amino Acid Sequence↗
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↗