Biomedical subjects

John Hesketh

Publications and source records attributed to John Hesketh.

9 recordsLinked to original sources

PubMed2006

Elongation factor 1alpha binds to the region of the metallothionein-1 mRNA implicated in perinuclear localization--importance of an internal stem-loop.

In eukaryotic cells, mRNA localization can provide local protein synthesis. Metallothionein-1 (MT-1) mRNA is associated with the perinuclear cytoskeleton, and this is essential for subsequent nuclear import of the protein. The present study defines the cis-acting localization signal and a trans-acting binding protein. Gel retardation and UV cross-linking assays using MT-1 3'UTR transcripts and CHO cell extracts revealed formation of a complex containing a approximately 50-kDa protein. Only localization-positive mutant transcripts competed for binding of this protein. Using an RNA affinity technique, Western blotting, mass spectrometry, and a supershift assay, the protein was identified as Elongation factor 1alpha (eEF1alpha). Mutation and deletion analysis showed that two regions, nucleotides 21-36 and 66-76, were required for both binding and localization. RNA-folding prediction combined with chemical and enzymatic probing experiments suggest that these regions are in juxtaposition within a stem/internal loop structure. Mutations that are predicted to alter this structure abrogate protein binding. Our hypothesis is that the cis-acting signal in MT-1 3'UTR is formed by this stem/internal loop, that it binds eEF1alpha, and that eEF1alpha-cytoskeleton interactions play a role in perinuclear mRNA localization.

3' Untranslated Regions↗

PubMed2006

Nutrient-gene interactions in benefit-risk analysis.

Individuals respond differently to nutrients and foods. This is reflected in different levels of benefits and risks at the same intake of a nutrient and, consequently, different 'windows of benefit' in terms of nutrient intake. This has led recently to the concept of 'personalised nutrition'. Genetic factors such as single nucleotide polymorphisms may be one source of this inter-individual variation in benefit-risk response to nutrients. In 2004 a European Union-funded network of excellence in the area of nutrigenomics (European Nutrigenomics Organisation; NuGO) organised a workshop on the role of nutrient-gene interactions in determining benefit-risk of nutrients and diet. The major issues discussed at the workshop are presented in the present paper and highlighted with examples from the presentations. The overall consensus was that although genetics provides a new vision where genetic information could in the future be used to provide knowledge on disease predisposition and nutritional requirements, such a goal is still far off and much more research is required before we can reliably include genetic factors in the risk-benefit assessment of nutrients and diets.

Diet↗

PubMed2005

An AU-rich stem-loop structure is a critical feature of the perinuclear localization signal of c-myc mRNA.

In eukaryotic cells, several mRNAs including those of c-myc and c-fos are localized to the perinuclear cytoplasm and associated with the cytoskeleton. The localization element of c-myc mRNA is present within its 3'UTR (3'-untranslated region) but the precise nature of this signal has remained unidentified. Chemical/enzymatic cleavage with RNases (ribonucleases) and lead have identified single-stranded and double-stranded regions in RNA transcripts of nucleotides 194-280 of the c-myc 3'UTR. Combined with computer predicted structure these results indicate that this region folds so that part of it forms a stem-loop structure. A mutation, that has been previously shown to prevent localization, leads to a different secondary RNA structure in this region as indicated by altered cleavage patterns. Competitive gel-retardation assays, using labelled transcripts corresponding to nucleotides 205-280 of c-myc 3'UTR, and fibroblast extracts revealed that the stem-loop region was sufficient for RNA-protein complex formation. In situ hybridization studies in cells transfected with reporter constructs, in which all or parts of the region corresponding to this stem-loop were linked to beta-globin, indicated that this region was sufficient for localization and that deletion of the nucleotides corresponding to the proposed upper-stem or terminal loop prevented localization. Our hypothesis is that an AU-rich stem-loop structure within nt 222-267 in the c-myc 3'UTR forms the perinuclear localization signal. Bioinformatic analysis suggests that this signal shares features with 3'UTRs of other localized mRNAs and that these features may represent a conserved form of signal in mRNA localization mechanisms.

3' Untranslated Regions↗

PubMed2005

An eleven nucleotide section of the 3'-untranslated region is required for perinuclear localization of rat metallothionein-1 mRNA.

Localization of mRNAs provides a novel mechanism for synthesis of proteins close to their site of function. MT1 (metallothionein-1) is a small, metal-binding protein that is largely cytoplasmic but which can be found in the nucleus. The localization of rat MT1 requires the perinuclear localization of its mRNA by a mechanism dependent on the 3'-UTR (3'-untranslated region). The present study investigates the nature of this mRNA localization signal using Chinese-hamster ovary cells transfected with gene constructs in which either MT1 or the globin coding region is linked to different sequences from the MT1 3'-UTR. Deletion, mutagenesis and antisense oligonucleotide approaches indicate that nt 45-76 of the 3'-UTR, in particular nt 66-76, are required for the localization of either MT1 mRNA or chimaeric transcripts in which a beta-globin coding region is linked to sequences from the MT1 3'-UTR. This section of the 3'-UTR contains a CACC repeat. Two mutations that are predicted to alter the secondary structure of this region also impair localization. Our hypothesis is that the perinuclear localization signal in MT1 mRNA is formed by a combination of the CACC repeat and its structural context.

3' Untranslated Regions↗

PubMed2005

Polarised distribution of the RNA-binding protein Staufen in differentiated intestinal epithelial cells.

mRNA localisation, as a mechanism for directing localised protein synthesis, plays a vital role in the functioning of certain cells, such as neurons and oocytes. Potentially this mechanism may also occur in polarised intestinal epithelial cells. Here we show that Staufen1(55), a protein involved in mRNA localisation and transport, is asymmetrically distributed in differentiated Caco-2 intestinal epithelial cells and partly co-localised with calnexin, a marker of the endoplasmic reticulum. The localisation to the apical region of the cell indicates that Staufen may be involved in localisation of transcripts to this domain.

Caco-2 Cells↗

PubMed2005

Effects of Se-depletion on glutathione peroxidase and selenoprotein W gene expression in the colon.

Selenium (Se)-containing proteins have important roles in protecting cells from oxidative damage. This work investigated the effects of Se-depletion on the expression of the genes encoding selenoproteins in colonic mucosa from rats fed diets of different Se content and in human intestinal Caco-2 cells grown in Se-adequate or Se-depleted culture medium. Se-depletion produced statistically significant (P<0.05) falls in glutathione peroxidase (GPX) 1 mRNA (60-83%) and selenoprotein W mRNA (73%) levels, a small but significant fall in GPX4 mRNA (17-25%) but no significant change in GPX2. The data show that SelW expression in the colon is highly sensitive to Se-depletion.

Animals↗

PubMed2005

Annexin A2 binds to the localization signal in the 3' untranslated region of c-myc mRNA.

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis-acting sequences in the 3' untranslated regions (3'UTRs) of the mRNAs concerned acting together with trans-acting proteins. The C-MYC transcription factor is a proto-oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c-myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3'UTR. Here we show the specific binding of a trans-acting factor to the perinuclear localization element in the 3'UTR of c-myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross-linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c-myc 3'UTR implicated in localization; a protein of approximately 36 kDa exhibited specific, Ca(2+)-dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA-affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA-protein complex formed by cell extracts was further retarded by anti-(annexin A2). Purified annexin A2 bound to the same region of the c-myc 3'UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA-binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.

3' Untranslated Regions↗

PubMed2004

Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores.

Under resting conditions, the endoplasmic reticulum (ER) intraluminal free calcium concentration ([Ca(2+)](ER)) reflects a balance between active uptake by Ca(2+)-ATPases and passive efflux via 'leak channels'. Despite their physiological importance and ubiquitous leak pathway mechanism, very little is known about the molecular nature of these channels. As it has been suggested that the open translocon pore complex of the ER is permeable to ions and neutral molecules, we hypothesized that the ribosome-bound translocon would be permeable to calcium after treatment with puromycin, a translation inhibitor that specifically releases polypeptide chains. At this time, the translocon channel is left open. We measured the fluctuations in cytoplasmic and luminal calcium concentrations using fluorescent dyes (fura-2 and magfura-2, respectively). The calcium release induced by thapsigargin (a Ca(2+)-ATPase inhibitor) was lower after puromycin treatment. Puromycin also reduced the [Ca(2+)](ER) level when perfused into the medium, but was ineffective after anisomycin pre-treatment (an inhibitor of the peptidyl transferase). Puromycin had a similar effect in the presence of heparin and ryanodine. This puromycin-evoked [Ca(2+)](ER) decrease was specific to the translocon. We conclude that the translocon complex is a major calcium leak channel. This work reveals a new role for the translocon which is involved in the control of the [Ca(2+)](ER) and could therefore supervise many physiological processes, including gene expression and apoptosis.

Calcium↗

PubMed2004

Zipcodes and postage stamps: mRNA localisation signals and their trans-acting binding proteins.

Messenger RNA (mRNA) localisation is a widespread mechanism within eukaryotic cells that provides local synthesis of proteins close to where they function. In general, this mRNA targeting involves the cytoskeleton and signals within the 3' untranslated region (3'UTR) of the transcript. In this paper, the authors review what is known of the nature of the localisation signals and the proteins that interact with them in animal cells. Specific examples are selected to illustrate the emerging pattern of how these signals are formed by the mRNA and the key RNA-binding proteins. The signals are usually restricted to relatively short regions of the 3'UTR, but their precise nature varies, with both sequence and structure playing key roles. Repeat motifs and functional redundancy also appear as common features of these signals. The trans-acting factors involved in localisation include proteins having other roles in nuclear events, proteins that shuttle between the nucleus and the cytoplasm and translational factors. In addition, there is evidence of homology among these proteins and the mechanisms of localisation across eukaryotic species.

3' Untranslated Regions↗