Prevention of type 2 diabetes. Health promotion helps no one.
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Activities of Daily Living↗
Biomedical subjects
C Guthrie
Publications and source records attributed to C Guthrie.
An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport.
Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery.
Active Transport, Cell Nucleus↗
Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase.
Mammalian U2AF65 and UAP56 are required for prespliceosome (PS) formation. We tested the predictions that the yeast UAP56 homolog, SUB2, is required for the same step and functions collaboratively with MUD2, the yeast homolog of U2AF65. Unexpectedly, sub2-1 extracts accumulate PS-like complexes. Moreover, deletion of MUD2 exacerbates the cs phenotype of sub2 alleles yet suppresses both the ts sub2-1 and the lethal Deltasub2 phenotypes. We propose that Sub2 functionally interacts with Mud2 both before and after PS formation. In the absence of Mud2, Sub2 function becomes dispensable.
Adenosine Triphosphatases↗
Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation.
Mammalian SR proteins are currently thought to function in mRNA export as well as splicing. They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides. Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear. Npl3 is a shuttling protein in budding yeast that we showed previously to be a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1. Here we demonstrate that Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p. The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo. We propose that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export.
Arginine↗
Food and activity preferences in children of lean and obese parents.
BACKGROUND: Children of obese parents have a substantially higher risk of adult obesity than children of lean parents. Adoption and twin studies have shown that this risk is largely genetic but the proximal mechanisms of the genetic risk are not known. Comparisons of energy intake or expenditure in children of obese and lean parents have produced mixed, but generally negative results. An alternative hypothesis is that the early expression of obesity risk is through food and activity preferences, which provides a basis for later weight gain. The aim of this study was therefore to compare food and activity preferences in a large sample of young children from obese and lean families using parental obesity as a marker of the obesity-risk phenotype. Because the children from the families with obese parents were not yet overweight, differences observed in the two types of families are more likely to be causes than effects of obesity. METHODS: A total of 428 children aged 4-5 y, whose parents were either obese/overweight or normal-weight/lean were selected from a population sample of families with twin births. Food and activity preferences were assessed with a combination of food intake and taste tasks, and questionnaires completed by the mother during a home visit. FINDINGS: Children from the obese/overweight families had a higher preference for fatty foods in a taste test, a lower liking for vegetables, and a more 'overeating-type' eating style. They also had a stronger preference for sedentary activities, and spent more time in sedentary pastimes. There were no differences in speed of eating or reported frequency of intake of high-fat foods. CONCLUSION: Part of the process whereby a genetic risk of obesity is transmitted to the next generation could be through differences in diet and activity preferences, which would place susceptible individuals at risk of positive energy balance in the permissive nutritional environment of industrialised countries today.
Child, Preschool↗
Genetic interactions between the 5' and 3' splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing.
The YAG/ consensus sequence at the 3' end of introns (the slash indicates the location of the 3' splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5' end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5' /GUA with Y-3 of the 3' YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5' /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y-3 in the 3' YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y-3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5' /GUA and the GAG of U6 function in binding the 3' YAG/ during the second catalytic step.
Base Sequence↗
A putative ubiquitin ligase required for efficient mRNA export differentially affects hnRNP transport.
BACKGROUND: In the nucleus, mRNAs are bound by hnRNP proteins. A subset of hnRNP proteins shuttle between the nucleus and cytoplasm and are believed to promote mRNA export by acting as adaptors between mRNA and the transport machinery. The existence of multiple shuttling hnRNP proteins raises the question of whether differentially regulated, hnRNP-specific mRNA export pathways exist. RESULTS: We have determined that Tom1p, a conserved protein with a hect (homology to E6-AP carboxyl terminus) E3 ubiquitin ligase domain, is required for efficient mRNA export in S. cerevisiae, yet differentially affects hnRNP protein localization and export. Mutations in tom1 predicted to abolish ubiquitin ligase activity block efficient export of Nab2p and mRNA, causing Nab2p-mRNA complexes to accumulate in a punctate pattern coincident with the nuclear pore complex (NPC). Notably, the subcellular distribution of several other hnRNP proteins is not affected. In particular, Np13p remains mRNA-associated and continues to be efficiently exported in tom1 mutants. CONCLUSION: Our results demonstrate that mutations predicted to affect the enzymatic activity of the Tom1p ubiquitin ligase differentially affect export of hnRNP proteins in association with mRNA. We propose the existence of multiple mRNA export pathways, with export of Nab2p-associated mRNAs dependent on a branch of the ubiquitin protein modification pathway.
Amino Acid Sequence↗
Childhood obesity. Opportunity for physical activity has been lost.
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Child↗
The question remains: is the spliceosome a ribozyme?
The two phosphoryl transfer steps of pre-mRNA splicing are catalyzed within the large ribonuclear protein machine called the spliceosome. The highly dynamic nature of the spliceosome has presented many challenges to a structural and mechanistic understanding of its catalytic core. While much evidence supports the popular hypothesis that the catalytic steps of pre-mRNA splicing are mediated by spliceosomal RNA, a role for protein in catalysis cannot yet be ruled out. A highly conserved protein, Prp8, is a component of the catalytic core. We review data consistent with the hypothesis that Prp8 functions as a cofactor to an RNA enzyme.
Catalysis↗
Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome.
The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing.
Alleles↗
Conservation in budding yeast of a kinase specific for SR splicing factors.
SR protein kinases (SRPKs) and their substrates, the SR family of serine/arginine-rich pre-mRNA splicing factors, appear to be key regulators of alternative splicing. Although SR proteins have been well characterized through biochemical experiments in metazoans, their functions in vivo are unclear. Because of the strict splice site consensus and near absence of alternative splicing in Saccharomyces cerevisiae, it had been thought that budding yeast would lack an SRPK and its substrates. Here, we present structural, biochemical, and cell-biological evidence that directly demonstrates an SR protein kinase, Sky1p, as well as a number of SRPK substrates in S. cerevisiae. One of these substrates is Npl3p, an SR-like protein involved in mRNA export. This finding raises the provocative possibility that Sky1p, and by extension metazoan SRPKs, regulates mRNA export or the nucleocytoplasmic shuttling of RS domain proteins. The unexpected discovery of an SR protein kinase in budding yeast provides a foundation for genetic dissection of the biological functions of SR proteins and their kinases.
Alternative Splicing↗
An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p.
Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA. By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core. Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch. In vitro, the switch requires both Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing.
Actins↗
Nurses' perceptions of sexuality relating to patient care.
This paper presents a research study which explores the perceptions of nurses regarding sexuality related to patient care. A qualitative approach was taken to the research. The methodology used was grounded theory. The findings illustrate that nurses had difficulty dealing with issues of sexuality, and the reasons for this were complex and interrelated. Patient care was influenced by nurses' perceptions of sexuality. Nurses in the study did not, generally, provide patient care relating to sexuality. Nurses were reluctant to discuss sexuality with their patients. Stereotyped images of nurses, males and homosexuals were common. Nurses dealt with issues of patient sexuality by employing a variety of coping strategies.
Adaptation, Psychological↗
RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2.
BACKGROUND: The dynamic rearrangements of RNA structure which occur during pre-mRNA splicing are thought to be mediated by members of the DExD/H-box family of RNA-dependent ATPases. Although three DExD/H-box splicing factors have recently been shown to unwind synthetic RNA duplexes in purified systems, in no case has the natural biological substrate been identified. A duplex RNA target of particular interest is the extensive base-pairing interaction between U4 and U6 small nuclear RNAs. Because these helices must be disrupted to activate the spliceosome for catalysis, this rearrangement is believed to be tightly regulated in vivo. RESULTS: We have immunopurified Brr2, a DEIH-box ATPase, in a native complex containing U1, U2, U5 and duplex U4/U6 small nuclear ribonucleoprotein particles (snRNPs). Addition of hydrolyzable ATP to this complex results in the disruption of U4/U6 base-pairing, and the release of free U4 and U6 snRNPs. A mutation in the helicase-like domain of Brr2 (brr2-1) prevents these RNA rearrangements. Notably, U4/U6 dissociation and release occur in the absence of exogenously added pre-mRNA. CONCLUSIONS: Disruption of U4/U6 base-pairing in native snRNPs requires ATP hydrolysis and Brr2. This is the first assignment of a DExD/H-box splicing factor to a specific biological unwinding event. The unwinding function of Brr2 can be antagonized by the annealing activity of Prp24. We propose the existence of a dynamic cycle, uncoupled from splicing, that interconverts free and base-paired U4/U6 snRNPs.
Adenosine Triphosphatases↗
The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro.
BACKGROUND: During pre-mRNA splicing, dynamic rearrangement of RNA secondary structure within the spliceosome is crucial for intron recognition and formation of the catalytic core. Splicing factors belonging to the DExD/DExH-box family of RNA-dependent ATPases are thought to have a central role in directing these rearrangements by unwinding RNA helices. Proof of this hypothesis has, however, been conspicuously lacking. RESULTS: Prp16 is a DEAH-box protein that functions in the second step of splicing in vitro. Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity. This activity is independent of sequence in either the single-stranded or duplexed regions of the RNA substrate. A mutation (prp16-1) near the ATP-binding motif of Prp16 inhibits both the RNA-dependent ATPase activity and the ATP-dependent RNA unwinding activity. CONCLUSIONS: Our findings provide strong biochemical evidence that Prp16 can disrupt a duplexed RNA structure on the spliceosome. Because the purified protein lacks sequence specificity in unwinding RNA duplexes, targeting of the unwinding activity of Prp16 in the spliceosome is likely to be determined by other interacting protein factors. The demonstration of unwinding activity will also help our understanding of how the fidelity of branchpoint recognition is controlled by Prp16.
Adenosine Triphosphatases↗
Mechanical devices of the spliceosome: motors, clocks, springs, and things.
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Adenosine Triphosphatases↗
A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles.
The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure. The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small nuclear RNAs (snRNAs). Here, the yeast RNA binding protein Prp24 is shown to reanneal these snRNAs. When Prp24 is absent, unpaired U4 and U6 small nuclear ribonucleoprotein particles (snRNPs) accumulate; with time, splicing becomes inhibited. Addition of purified Prp24 protein regenerates duplex U4/U6 snRNPs for new rounds of splicing. The reannealing reaction catalyzed by Prp24 proceeds more efficiently with snRNPs than with deproteinized snRNAs.
Adenosine Triphosphate↗
PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain.
Dynamic rearrangement of RNA structure is crucial for intron recognition and formation of the catalytic core during pre-mRNA splicing. Three of the splicing factors that contain sequence motifs characteristic of the DExD/DExH-box family of RNA-dependent ATPases (Prp16, Prp22, and the human homologue of Brr2) recently have been shown to unwind RNA duplexes in vitro, providing biochemical evidence that they may direct structural rearrangements on the spliceosome. Notably, however, the unwinding activity of these proteins is sequence nonspecific, raising the question of how their functional specificity is determined. Because the highly conserved DExD/DExH-box domain in these proteins is typically flanked by one or more nonconserved domains, we have tested the hypothesis that the nonconserved regions of Prp16 determine the functional specificity of the protein. We found that the nonconserved N-terminal domain of Prp16 is (1) essential for viability, (2) required for the nuclear localization of Prp16, and (3) capable of binding to the spliceosome specifically at the step of Prp16 function. Moreover, this domain can interact with the rest of the protein to allow trans-complementation. Based on these results, we propose that the spliceosomal target of the unwinding activity of Prp16, and possibly other DExD/DExH-box splicing factors as well, is defined by factors that specifically interact with the nonconserved domains of the protein.
Adenosine Triphosphatases↗