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Biomedical subjects

J E McCarthy

Publications and source records attributed to J E McCarthy.

At least 19 recordsLinked to original sources
PubMed2001

A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties.

The eukaryotic cap-binding proteins belonging to the eIF4E family are generally involved in mediating the recruitment of ribosomes to capped mRNA. We described previously a cap-binding protein (now called eIF4E1) in Schizosaccharomyces pombe that appears to have all of the usual structural and functional attributes of an eIF4E. We have now characterised a new type of cap-binding protein (eIF4E2) from this organism, which at the amino acid sequence level, is 52% identical and 59% similar to eIF4E1. eIF4E2 is not essential in S.pombe but has some novel properties that may be related to a special function in the cell. The ratio of eIF4E2:eIF4E1 in the cell shifts in favour of eIF4E2 at higher temperatures. Despite having all of the dorsal face amino acids that have so far been associated with eIF4G binding to eIF4E1, eIF4E2 binds the eIF4E-binding domain of S.pombe eIF4G >10(2)-times weaker than eIF4E1 in vitro. The eIF4E2 cap-binding affinity is in the typical micromolar range. The results suggest that eIF4E2 is not active on the main pathway of translation initiation in fission yeast but might play a role in the adaptation strategy of this organism under specific growth conditions. Moreover, they provide insight into the molecular characteristics required for tight binding to eIF4G.

5' Untranslated Regions↗
PubMed2001

A quantitative molecular model for modulation of mammalian translation by the eIF4E-binding protein 1.

Translation initiation is a key point of regulation in eukaryotic gene expression. 4E-binding proteins (4E-BPs) inhibit initiation by blocking the association of eIF4E with eIF4G, two integral components of the mRNA cap-binding complex. Phosphorylation of 4E-BP1 reduces its ability to bind to eIF4E and thereby to compete with eIF4G. A novel combination of biophysical and biochemical tools was used to measure the impact of phosphorylation and acidic side chain substitution at each potentially modulatory site in 4E-BP1. For each individual site, we have analyzed the effects of modification on eIF4E binding using affinity chromatography and surface plasmon resonance analysis, and on the regulatory function of the 4E-BP1 protein using a yeast in vivo model system and a mammalian in vitro translation assay. We find that modifications at the two sites immediately flanking the eIF4E-binding domain, Thr(46) and Ser(65), consistently have the most significant effects, and that phosphorylation of Ser(65) causes the greatest reduction in binding affinity. These results establish a quantitative framework that should contribute to understanding of the molecular interactions underlying 4E-BP1-mediated translational regulation.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The apical angle: a mathematical analysis of the ellipse.

BACKGROUND: The elliptical excision is a common surgical procedure. The dermatologic literature predominantly describes an excisional geometry with a 3:1 length:width ratio and an apical angle of 30 degrees. OBJECTIVE: To analyze the elliptical excision by applying mathematical principles and define the apical angle and its relationship to the length:width ratio. METHODS: We examined numerous examples of elliptical excisions as presented in the dermatologic literature. We analyzed the geometry of the excisions and defined it mathematically. RESULTS: The apical angle of a 3:1 elliptical excision is not 30 degrees. The true apical angle varies from 37 degrees to 74 degrees depending on excisional geometry. CONCLUSION: The commonly presented apical angle of 30 degrees is incorrect and does not reflect the true apical angle of elliptical excisions.

Dermatologic Surgical Procedures↗
PubMed2000

Translation initiation: insect virus RNAs rewrite the rule book.

Picorna-like insect virus RNAs direct an unorthodox form of translation initiation at a non-AUG-related codon, without involvement of initiator tRNA. This seems to involve a special type of mRNA pseudoknot structure which allows bypassing of the usual P-site-dependent mechanism.

Animals↗
PubMed2000

Stabilization of eukaryotic initiation factor 4E binding to the mRNA 5'-Cap by domains of eIF4G.

The eukaryotic cap-binding complex eIF4F is an essential component of the translational machinery. Recognition of the mRNA cap structure through its subunit eIF4E is a requirement for the recruitment of other translation initiation factors to the mRNA 5'-end and thereby for the attachment of the 40 S ribosomal subunit. In this study, we have investigated the mechanistic basis of the observation that eIF4E binding to the cap is enhanced in the presence of the large eIF4F subunit, eIF4G. We show that eIF4E requires access to both the mRNA 5'-cap and eIF4G to form stable complexes with short RNAs. This stabilization can be achieved using fragments of eIF4G that contain the eIF4E binding site but not the RNA recognition motifs. Full-length eIF4G is shown to induce increased eIF4E binding to cap analogues that do not contain an RNA body. Both results show that interaction of eIF4G with the mRNA is not necessary to enhance cap binding by eIF4E. Moreover, we show that the effect of binding of full-length eIF4G on the cap affinity of eIF4E can be further modulated through binding of Pab1 to eIF4G. These data are consistent with a model in which heterotropic cooperativity underlies eIF4F function.

Chromatography, Affinity↗
PubMed2000

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex.

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.

Blotting, Western↗
PubMed2000

Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination.

The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.

3' Untranslated Regions↗
PubMed1999

Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state.

Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Post-termination ribosome interactions with the 5'UTR modulate yeast mRNA stability.

A novel form of post-transcriptional control is described. The 5' untranslated region (5'UTR) of the Saccharomyces cerevisiae gene encoding the AP1-like transcription factor Yap2 contains two upstream open reading frames (uORF1 and uORF2). The YAP2-type of uORF functions as a cis-acting element that attenuates gene expression at the level of mRNA turnover via termination-dependent decay. Release of post-termination ribosomes from the YAP2 5'UTR causes accelerated decay which is largely independent of the termination modulator gene UPF1. Both of the YAP2 uORFs contribute to the destabilization effect. A G/C-rich stop codon context, which seems to promote ribosome release, allows an uORF to act as a transferable 5'UTR-destabilizing element. Moreover, termination-dependent destabilization is potentiated by stable secondary structure 3' of the uORF stop codon. The potentiation of uORF-mediated destabilization is eliminated if the secondary structure is located further downstream of the uORF, and is also influenced by a modulatory mechanism involving eIF2. Destabilization is therefore linked to the kinetics of acquisition of reinitiation-competence by post-termination ribosomes in the 5'UTR. Our data explain the destabilizing properties of YAP2-type uORFs and also support a more general model for the mode of action of other known uORFs, such as those in the GCN4 mRNA.

5' Untranslated Regions↗
PubMed1999

Translational repression by human 4E-BP1 in yeast specifically requires human eIF4E as target.

4E-binding proteins (4E-BPs) are believed to have important regulatory functions in controlling the rate of translation initiation in mammalian cells. They do so by binding to the mRNA cap-binding protein, eIF4E, thereby inhibiting formation of the cap-binding complex, a process essential for cap-dependent translation initiation. We have reproduced the translation-repressive function of human 4E-BP1 in yeast and find its activity to be dependent on substitution of human eIF4E for its yeast counterpart. Translation initiation and growth are inhibited when human 4E-BP1 is expressed in a strain with the human eIF4E substitution, but not in an unmodified strain. We have compared the relative affinities of human 4E-BP1 for human and yeast eIF4E, both in vitro using an m7GTP cap-binding assay and in vivo using a yeast two-hybrid assay, and find that the affinity of human 4E-BP1 for human eIF4E is markedly greater than for yeast eIF4E. Thus yeast eIF4E lacks structural features required for binding to human 4E-BP1. These results therefore demonstrate that the features of eIF4E required for binding to 4E-BP1 are distinct from those required for cap-complex assembly.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A systematic nomenclature for new translation initiation factor genes from S. pombe and other fungi.

Eukaryotic translation initiation factors and their corresponding genes have been characterized using biochemical and genetic methods from a variety of different organisms. The designations of the factors relate to their apparent roles in the biochemical process. Many gene names indicate genetic interactions with other genes or the functional attributes used to identify them. On the other hand, progress in systematic sequencing of the genomes of organisms like Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes homologous to known translation initiation factor genes. The genes defined by the systematic sequencing approach are assigned numerical designations completely unrelated to their biological function. So far there have been publications on only three genes encoding translation initiation factors from Schizosaccharomyces pombe. We therefore see this an an ideal opportunity to propose a systematic and logical nomenclature for genes encoding translation initiation factor genes that can be applied to all further genes of this type that are characterized in this fission yeast.

Fungi↗
PubMed1999

Outcome predictors in pregnant opiate and polydrug users.

A retrospective case note study of 93 women was performed in order to assess the effect of maternal factors on neonatal outcome in a group of women attending a specialist clinic for pregnant drug users. There were no significant differences in outcome for chaotic drug users compared with non-chaotic drug users, or for cocaine users compared with non-cocaine using drug users. Women who reduced their methadone dose during pregnancy delivered babies of significantly higher birth weight than those whose methadone dose remained the same or increased (median 3027 g, range 1780-3629 g vs 2645 g, range 580-3720 g). Women who abused benzodiazepines during pregnancy produced babies of significantly lower birth weight than those women who did not use benzodiazepines (median 2100 g, range 580-3520 g vs 2767 g, range 1530-3720 g). The results of this study give healthcare staff evidence to use in encouraging drug-using women to avoid benzodiazepines during pregnancy and to reduce their methadone dosage. The treatment received from a specialist clinic may mitigate against some of the other recognised effects of drug use during pregnancy.

Benzodiazepines↗
PubMed1998

Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae.

A lipase from Rhizopus oryzae DSM 853 (ROL) was cloned from a chromosomal gene bank, sequenced and overexpressed in Escherichia coli. ROL and its precursors ProROL and PreProROL were purified and their pH and temperature profile was determined. In contrast to ROL, ProROL and PreProROL had considerably higher thermostability and a slightly higher pH optimum. Moreover, it could be demonstrated by in vitro experiments that the natural leader sequence of ROL is able to inhibit the folding supporting properties of the prosequence, resulting in a retardation of folding. In addition, there is strong evidence that all different lipase forms derived from Rhizopus sp. described in the literature are a result of different proteolytic processing and originate from the same gene.

Amino Acid Sequence↗
PubMed1998

Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.

Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

Amino Acid Sequence↗
PubMed1998

High-energy magnetic Compton scattering experiments at ESRF.

Investigations of spin densities in ferromagnetic materials using magnetic Compton scattering are reported. At the high-energy beamline ID15 at the ESRF, experiments have been carried out utilizing the high flux at very high photon energies. Energies from 60 up to 1000 keV have been used for investigations of experimental resolution, cross section, spin moments and momentum distribution. Optimized conditions are found for photon energies from 200 to 250 keV with a momentum resolution < 0.4 a.u. and a doubled magnetic effect compared with earlier measurements. In the determination of absolute spin moments multiple scattering has to be taken into account.

Journal Article↗
PubMed1998

The yeast transcription factor genes YAP1 and YAP2 are subject to differential control at the levels of both translation and mRNA stability.

Two forms of post-transcriptional control direct differential expression of the Saccharomyces cerevisiae genes encoding the AP1-like transcription factors Yap1p and Yap2p. The mRNAs of these genes contain respectively one (YAP1 uORF) and two (YAP2 uORF1 and uORF2) upstream open reading frames. uORF-mediated modulation of post-termination events on the 5'-untranslated region (5'-UTR) directs differential control not only of translation but also of mRNA decay. Translational control is defined by two types of uORF function. The YAP1 -type uORF allows scanning 40S subunits to proceed via leaky scanning and re-initiation to the major ORF, whereas the YAP2 -type acts to block ribosomal scanning by promoting efficient termination. At the same time, the YAP2 uORFs define a new type of mRNA destabilizing element. Both post-termination ribosome scanning behaviour and mRNA decay are influenced by the coding sequence and mRNA context of the respective uORFs, including downstream elements. Our data indicate that release of post-termination ribosomes promotes largely upf -independent accelerated decay. It follows that translational termination on the 5'-UTR of a mature, non-aberrant yeast mRNA can trigger destabilization via a different pathway to that used to rid the cell of mRNAs containing premature stop codons. This route of control of non-aberrant mRNA decay influences the stress response in yeast. It is also potentially relevant to expression of the sizable number of eukaryotic mRNAs that are now recognized to contain uORFs.

Base Sequence↗
PubMed1998

Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster.

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

Amino Acid Sequence↗