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Localisation of a reporter transcript by the c-myc 3[prime]-UTR is linked to translation
Introduction
Materials And Methods
DNA constructs
Cell culture and transfection
Translation analysis
In situ hybridisation
Results And Discussion
Desferrioxamine reduces translation of chimeric transcripts containing an IRE
Localisation of IRE-globin-myc transcripts
Stability of IRE-globin-myc transcripts
Conclusions
Acknowledgements
References
Localisation of a reporter transcript by the c-myc 3[prime]-UTR is linked to translation
Received August 16, 1999; Revised September 20, 1999; Accepted September 27, 1999
ABSTRACT The 3[prime]-untranslated region of c-myc mRNA contains a perinuclear localisation signal which is sufficient to target [beta]-globin coding sequences. The link between perinuclear mRNA localisation and translation has been investigated using cells transfected with chimeric gene constructs in which globin reporter sequences were linked to the c-myc 3[prime]-untranslated region and the iron-responsive element from ferritin mRNA. Iron supplementation of the medium promoted translation of the chimeric mRNA as assessed by its presence in polysomes; in situ hybridisation showed that the mRNA was localised around the nucleus. Treatment with the iron chelator desferrioxamine for 16 h prevented both translation and mRNA localisation. In controls where the expressed mRNA lacked the iron-responsive element desferrioxamine had no effect upon localisation. In contrast, arrest of on-going global translation by puromycin treatment had no effect on mRNA localisation. The data suggest that if initiation of translation of a mRNA containing the c-myc localisation signal is prevented in some way then localisation does not occur, whereas once the mRNA has been localised further translation is not required to maintain mRNA localisation.
INTRODUCTION
Cell organisation requires that newly synthesised proteins are delivered to their site of function. Protein targeting by motifs within the protein structure plays an important role in this process; for example, import of proteins into the nucleus requires a nuclear localisation signal (1). Recently, some mRNAs have been found to be localised in mammalian cells (2-4) and this localisation has the potential to contribute to protein delivery by allowing synthesis of proteins close to their site of function. In those cases so far studied in detail, the mRNA localisation signal occurs in the 3[prime]-untranslated region (3[prime]-UTR) (2-4).
The mRNA encoding the nuclear transcription factor c-myc has been found to be associated with the cytoskeleton (5) and to be localised in the perinuclear cytoplasm (6). This association and localisation requires 3[prime]-UTR sequences; furthermore, the c-myc 3[prime]-UTR is capable of targeting a [beta]-globin reporter to the perinuclear cytoplasm (5-7). Since both c-myc mRNA and c-myc protein are unstable, the significance of the perinuclear localisation of the mRNA may be to facilitate efficient import of the newly synthesised protein into the nucleus. In contrast, signals in the 3[prime]-UTRs of actin and myelin basic protein mRNAs are sufficient to allow transport of a reporter to the cell periphery or down cell processes (8,9).
In a small number of cases it has been shown that proteins bind to the 3[prime]-UTR localisation element (10-12). However, the sequence of biochemical events that occur during localisation and translation of mRNAs has not been defined and the potential links between translation and localisation have not been investigated in detail. The observation that myelin basic protein mRNA is transported in granules which also contain ribosomal components and amino acid synthetases (13) indicates that mRNAs may be localised in some form of complex with other translation factors.
In the present experiments we have used two different approaches to investigate if localisation of specific transcripts is perturbed by either prevention of formation of a translation complex or dissociation of the complex once translation is occurring. Firstly, cells transfected with chimeric gene constructs containing an iron-responsive element (IRE) were used to ascertain whether the prevention of formation of a translation complex interferes with such perinuclear localisation. The translation of ferritin mRNA is regulated by iron concentration through binding of a protein to a specific sequence/structure in the mRNA (14). Incorporation of this sequence into heterologous gene constructs produces chimeric transcripts whose translation is also regulated by iron (15). In the present work we have incorporated an IRE into a globin-myc gene construct, expressed these constructs in stable transfected cells and assessed localisation of the reporter transcripts by in situ hybridisation. Secondly, inhibition with puromycin has been used to study if on-going translation is required for perinuclear localisation of such globin reporter transcripts by the c-myc 3[prime]-UTR localisation signal; earlier studies have shown that in the case of [beta]-actin mRNA release of translational components from polysomes with puromycin does not affect localisation in the peripheral cytoplasm (16).
MATERIALS AND METHODS
DNA constructs
The construction of pGlobin-myc has been described previously (7). p[beta]IREGlob-myc was constructed by ligating the rabbit [beta]-globin XbaI-XhoI genomic fragment with the XhoI-KpnI mouse c-myc 3[prime]-UTR and flanking genomic sequence, and inserting the resulting fragment into the SmaI and KpnI sites of p[beta]IRE (15). This produced a construct in which the globin coding sequences were flanked by the IRE from ferritin at the 5[prime]-end and the c-myc 3[prime]-UTR at the 3[prime]-end. The XhoI-KpnI subfragment from p[beta]IREGlob-myc was then inserted into the EcoRI and KpnI sites of p[beta]IRE, followed by the BamHI-SacI luciferase-containing fragment from pGEM-luc (Promega) into the same sites, to give rise to p[beta]IRELuc-3[prime]myc.
Cell culture and transfection
Cells were grown in 90 mm Petri dishes for mRNA stability studies and in glass chamber slides for in situ hybridisation. Ltk- fibroblasts were grown in Dulbecco's minimal Eagle's medium (Gibco BRL) and CHO cells in Ham's F12 modified with L-glutamine (ICN Biomedicals Inc.), both supplemented with 10% foetal calf serum, and in an atmosphere of 5% CO2. Cells were incubated with 10-100 µM ferric ammonium citrate or 100-300 µM desferrioxamine mesylate (Sigma) for 16 h prior to analysis (15).
Transfection of Ltk- fibroblasts was carried out using calcium phosphate and that of Chinese hamster ovary cells with lipofectamine. Cells were co-transfected with both the plasmid DNA of interest and a plasmid carrying neomycin resistance (6,14). Stable transfectants were selected by culture in the presence of 1 mg/ml G418. In addition, for certain translation studies the fibroblasts were transiently transfected with gene constructs expressing luciferase. In this case 104 cells were transfected with 1 µg of p[beta]IRELuc-3[prime]myc, 10 ng pCMV-RLuc (Promega) together with 1 µg carrier DNA and 10 µg lipofectamine (Gibco BRL).
Translation analysis
For polysome analysis cells were treated for 30 min with 100 µg/ml cycloheximide and then lysed as previously described (15) except that a syringe equipped with a 20G needle was used instead of a Dounce homogeniser. After an initial 15 min centrifugation at 10 000 g, cleared supernatants were centrifuged for 160 min at 40 000 r.p.m. through a 15-50% sucrose gradient in a SW41Ti Beckman rotor and collected in 0.5 ml fractions which were treated for 30 min at 37°C with 10 µg proteinase K in the presence of 2% SDS. RNA was recovered by solvent extraction and ethanol precipitation, separated by electrophoresis in a 1.5% agarose-formaldehyde denaturing gel, and analysed by northern hybridisation. The 32P-labelled rat GAPDH, rabbit [beta]-globin and human ferritin H chain cDNAs were sequentially used to probe the membranes.
Luciferase activity was measured following cell lysis according to the supplier's recommendations using the Dual-luciferaseTM reporter assay from Promega.
In situ hybridisation
Comparison of mRNA distribution was carried out in cells grown in multiwell chamber slides so that the different cell lines and different treatments to be studied could be grown in adjacent wells and all treatments and reactions carried out under identical conditions for a given set of cell lines: in this way mRNA distribution and its quantification was directly comparable. Fixation, hybridisation, detection and analysis were carried out using procedures described previously (6,7). Cells were washed three times with PBS prior to fixation for 10 min with 4% paraformaldehyde in PBS. The cells were partially dehydrated in 70% ethanol for 30 min and then fixed for a further 10 min in 4% paraformaldehyde, 0.2% Triton X-100 in PBS. After a further brief fixation for 5 min and two further washes in PBS, cells were incubated in 50% formamide, 2× SSC at room temperature before being hybridised overnight at 55°C with 200 ng of digoxigenin-labelled antisense riboprobe. The probe was generated using T7 polymerase from a 90 bp ApaI-EcoRI fragment of the last exon of the rabbit [beta]-globin gene using a DIG RNA labelling kit (Roche Molecular Biochemical, UK). Controls were either hybridised with a digoxigenin-labelled sense probe generated from the same fragment using SP6 polymerase or incubated with hybridisation mix containing no probe. After hybridisation cells were washed in 5× SSC for 30 min and then 50% formamide, 2× SSC, both at 55°C. Non-specifically bound probe was removed by treatment with 20 µg/ml RNase A in wash buffer (10 mM Tris-HCl, pH 7.5, containing 0.4 M NaCl and 5 mM EDTA) at 37°C. After a brief wash in wash buffer and two further washes in 2× SSC the bound probe was detected by incubation with alkaline phosphatase-linked anti-digoxigenin and incubation with 4-nitroblue tetrazolium for 16 h. The staining produced by the alkaline phosphatase activity was quantified using an image analysis system in which the images were captured using a Pulnix camera and Fenestra/Cyclops software (Kinetic Imaging Ltd, Liverpool, UK). Staining intensities were measured in perinuclear areas and adjacent areas of cytoplasm in the cell periphery. Three to four measurements were made in each cell and 30 individual cells selected at random were analysed for each cell line: the experiments and analyses were repeated at least three times. Values were corrected for staining blanks from cell-free areas.
RESULTS AND DISCUSSION
Desferrioxamine reduces translation of chimeric transcripts containing an IRE
Ltk- fibroblasts were transfected with the chimeric construct containing the IRE in a 5[prime] position to the globin coding region and the c-myc 3[prime]-UTR (IRE-GM). Cells were treated with either ferric ammonium chloride or the iron chelator desferrioxamine and cell extracts subjected to sucrose gradient separation and fractions analysed both for globin and ferritin mRNAs and for rRNA. As shown in Figure 1A, after addition of iron both globin and ferritin mRNAs were found in the heavier fractions in the middle of the gradient whereas after desferrioxamine treatment these two mRNAs were recovered in light fractions towards the top of the gradient. The position in the gradients of these lighter fractions coincided with the migration of ribosomal subunits released by EDTA treatment (Fig. 1B) and lighter ribonucleoprotein material. This indicated that desferrioxamine had prevented translation of both ferritin and IRE-GM transcripts. Cells treated with desferrioxamine showed similar rRNA profiles to cells supplemented with iron. In both cases the bulk of the RNA was recovered from the polysome-containing fractions in the middle of the gradient, indicating that desferrioxamine did not perturb overall protein synthesis. Thus, on the basis of presence in polysomes, desferrioxamine specifically reduces translation of ferritin (as shown previously; 14) and the IRE-GM transcripts. The ability of the IRE to regulate translation of a chimeric transcript was directly demonstrated in cells transfected with an IRE-luciferase-c-myc 3[prime]-UTR construct. These cells were grown in normal medium, in medium supplemented with iron to maximise translation of the transgene, or in the presence of desferrioxamine. The amount of luciferase derived from the transgene was similar in cells grown in normal or supplemented medium but was reduced in the presence of desferrioxamine (Fig. 1C), as was the level of endogenous ferritin (results not shown). Overall the data show that addition of the iron chelator desferrioxamine prevents translation of the IRE-GM mRNA. Thus, as previously found with chimeric globin-fos constructs (15), the translation of a heterologous mRNA containing an IRE is sensitive to the iron concentration the culture medium.
A
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B
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C
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Figure 1. The effect of modulating intracellular iron concentration on translation of a p[beta]IRELuc-3[prime]myc reporter and globin-myc transcripts containing an IRE. Polysomes were fractionated by centrifugation through sucrose gradients as described in Materials and Methods. The presence of [beta]-globin chimeric mRNA and endogenous ferritin mRNA was monitored by northern blotting and the autoradiograms quantified with a Millipore Bioscan image analyser. (A) Extracts from cells treated with 100 µM ferric ammonium citrate (open bars) or 300 µM desferrioxamine (closed bars) were analysed for globin (a) and ferritin (b) mRNAs. Ribosome distributions from cells treated with 100 µM ferric ammonium citrate (+FER) or 300 µM desferrioxamine (-FER) are shown below by the ethidium bromide staining pattern in the gels prior to transfer. (B) Extracts from control cells (open bars, -EDTA) or EDTA-treated (30 mM) cells (closed bars, +EDTA) were analysed by northern blotting and were successively probed with globin (c), ferritin (d) or GAPDH (not shown). The latter mRNA, which is insensitive to iron regulation, was used as an internal standard to correct for slight variations in the slopes of the different gradients. Ribosome distribution is shown below by the ethidium bromide staining pattern in the gels prior to transfer. (C) p[beta]IRELuc-3[prime]myc directing firefly luciferase expression was transiently transfected into Ltk- fibroblasts together with a renilia luciferase expression vector insensitive to iron modulation. After treatment for 16 h with 100 µM ferric ammonium citrate (+Fer) or 300 µM desferrioxamine (-Fer) the ratio of firefly to renilia luciferase activities was monitored and plotted as arbitrary units. The data shows >70% inhibition of luciferase activity after desferrioxamine treatment relative to iron treatment. This is consistent with the almost complete inhibition of ferritin accumulation observed by western blottting after the same treatment (data not shown). Luciferase activity was similar in cells without treatment (NT) and those treated with ferric ammonium citrate.
Localisation of IRE-globin-myc transcripts
Initially, mRNA localisation studies were carried out in Ltk- fibroblasts transfected with the same chimeric globin-myc constructs. In situ hybridisation to detect globin transcripts showed that under normal culture conditions there was strong staining around the nucleus of IRE-GM cells but little or no staining towards the cell periphery (Fig. 2A). The pattern of staining was essentially the same as that observed previously with GM cells (6,7), indicating that addition of the IRE has no intrinsic effect on localisation in cells grown in standard growth medium with adequate iron concentrations. When the transfected Ltk- fibroblasts were cultured with elevated Fe concentrations in the growth medium, conditions under which the chimeric globin transcripts are most efficiently translated, the perinuclear localisation of the mRNA was maintained (Fig. 2B). In contrast, addition of desferrioxamine for 16 h, which reduces translation of the globin transcripts, led to the perinuclear localisation of the globin transcripts being considerably less marked and strong staining was observed throughout the cytoplasm to the cell periphery (Fig. 2C). The distribution of transcripts in stable transfected Ltk- fibroblasts was further analysed by quantification of the staining pattern and image analysis (7). For each cell, staining intensity was measured in three small areas of identical size as described previously: first, intensity was measured in an area of the perinuclear cytoplasm chosen at random and defined as a part of cytoplasm close to the nucleus but not including any of it; next, the intensity was measured in an adjacent area of cytoplasm but in the cell periphery close to the cell membrane; finally, a cell-free blank measurement was taken. For a given cell three measurements were routinely taken in each area; staining intensity was highly consistent within an area of a single cell. Previous studies have shown such measurements to have a standard error of 3-8% (7). After subtraction of the blank, a perinuclear/peripheral staining ratio was calculated in order to give a quantitative and non-subjective measure of the degree to which there was perinuclear localisation of the transcripts in that cell. As shown in Figure 3, quantification of the staining pattern in the transfected Ltk- fibroblasts confirmed that in the IRE-GM cells reduced translation in the presence of desferrioxamine was associated with reduced perinuclear mRNA localisation. This effect was not seen with GM transcripts (no IRE present) showing that the effect required the presence of the IRE element (Fig. 3).
Figure 2. In situ hybridisation showing the effects of modulating iron concentration on globin transcript localisation in transfected fibroblasts. Ltk- fibroblasts transfected with [beta]IREGlob-myc were either cultured in normal medium (a), supplemented with 10 µM ferric ammonium citrate (b) or treated with 100 µM desferrioxamine for 16 h (c) prior to fixation and in situ hybridisation using an antisense riboprobe specific for globin coding sequences. Specific labelling was detected using alkaline phosphatase-linked anti-digoxigenin antibody and 4-nitroblue tetrazolium as substrate. Note the strong ring of staining around the nucleus in (a) and (b) but not in (c) where perinuclear staining was not apparent and the mRNA was present throughout the cytoplasm. Controls treated with a sense riboprobe (d) showed no staining. Scale bar 10 µm.
Figure 3. Quantification of the distribution of globin transcripts detected in transfected Ltk- fibroblasts by in situ hybridisation. Ltk- fibroblasts transfected with either Glob-myc (GM) or [beta]IREGlob-myc (GM-IRE) were cultured in normal medium (GM), supplemented with 10 µM ferric ammonium citrate (+ FE), treated with 100 µM desferrioxamine for 16 h (+ chelator), or treated with puromycin for 30 min prior to fixation and in situ hybridisation using an antisense riboprobe specific for globin coding sequences. Specific labelling was detected using alkaline phosphatase-linked anti-digoxigenin antibody and 4-nitroblue tetrazolium as substrate. Images were captured and staining was quantified in the perinuclear and peripheral cytoplasm of at least 30 cells chosen at random using Cyclops software. The ratio of perinuclear (pn) to peripheral (pf) staining was calculated (pn/pf). The data were collected from several experiments on the stable cell lines illustrated in Figure 2.
The effects of Fe and desferrioxamine on localisation were confirmed in Chinese hamster ovary cells expressing either globin-myc or IRE-GM transcripts (Fig. 4). These cells possess a different morphology from Ltk- fibroblasts, with a wider, more spread shape; again, the presence of desferrioxamine led to reduced perinuclear localisation of IRE-GM transcripts but not globin-myc transcripts without an IRE. GM and IRE-GM transcripts both show the same localisation (Figs 3 and 4), indicating that the presence of an IRE and its association with IRE-binding proteins does not influence localisation. Furthermore, addition of the IRE to globin-globin transcripts (and thereafter expression in the presence of iron or desferrioxamine) had no effect on the normal delocalised distribution of these transcripts (results not shown), indicating that interaction of the IRE with binding proteins does not itself bring about localisation. For these reasons it is most unlikely that the effects of desferrioxamine on localisation are secondary to relocalisation of IRE-binding proteins and the most likely explanation of the present data is that the modulation of translation initiation by desferrioxamine (see Fig. 1; 14) reduces mRNA localisation.
Figure 4. In situ hybridisation showing the effects of either arresting translation with puromycin or modulating iron concentration on globin transcript localisation in transfected CHO cells. CHO cells transfected with either pSVglob-myc (a and b) or p[beta]IREGlob-myc (c-f) were either cultured in normal medium (a and c), treated with puromycin for 30 min (b and d), supplemented with 10 µM ferric ammonium citrate (e) or treated with 100 µM desferroxiamine (f) prior to fixation and in situ hybridisation using an antisense riboprobe specific for globin coding sequences. Specific labelling was detected using alkaline phosphatase-linked anti-digoxigenin antibody and 4-nitroblue tetrazolium as substrate. Note the strong ring of staining around the nucleus in both GM and IRE-GM cells under control conditions and after puromycin treatment (compare a with b, and c with d). Perinuclear staining was still apparent in IRE-GM cells after growth in elevated iron concentrations (e) but not after chelation of the iron with desferrioxamine (f) when mRNA was present throughout the cytoplasm. Scale bar 10 µm.
It has been found previously that [beta]-actin mRNA localisation is unaffected by inhibition of all cellular translation by puromycin (16). As shown in Figures 3 and 4, in the present experiments addition of puromycin had no effect on the distribution of GM or IRE-GM transcripts.
Thus, modulation of translation of IRE-GM transcripts by puromycin or by desferrioxamine produces different results. The reason for this probably lies in the different mechanisms of action of the two compounds. Puromycin inhibits on-going translation within 30 min by dissociation of the polyribosome complex; since IRE-GM transcripts have a half-life of ~8 h, the in situ hybridisation analysis will detect transcripts which have been undergoing translation and then released from the polyribosomes by puromycin. In contrast, desferrioxamine chelates iron and when an IRE is present in a mRNA this will prevent initiation of translation (14). In the present experiments desferrioxamine was added 16 h (twice the half-life of the IRE-GM transcripts) before analysis. During this period on-going translation of the IRE-GM will have finished and ~75% of those translated transcripts degraded; meanwhile, further initiation will have been prevented by desferrioxamine (as shown in Fig. 1). Under these conditions the IRE-GM transcripts detected by in situ hybridisation will be largely those which have been prevented from being translated.
In contrast to the lack of effect of puromycin on [beta]-actin (16) and globin-myc localisation (Figs 3 and 4), it has recently been reported that in neurons total inhibition of protein synthesis by cycloheximide or puromycin causes ferritin mRNA to be no longer restricted in distribution to the cell body (17). There are several potential explanations for this difference. Firstly, retention of the ferritin mRNA in the neuronal cell body may involve a mechanism distinct from the localisation of actin and myc mRNAs by the cytoskeleton and signals within the 3[prime]-UTR (7,8); indeed, in liver, ferritin mRNA is partly cytosolic and partly found on the rough endoplasmic reticulum (18), and any mRNA associated with the endoplasmic reticulum would be released by puromycin. Alternatively, inhibition of total protein synthesis for 16 h may reduce levels of any number of relatively short-lived proteins and the low levels of such proteins, if they were involved in mRNA localisation or translation processes, could have secondary effects on localisation. Lastly, the inhibition of global cell protein synthesis, rather than the specific modulation of IRE-containing transcripts as in the present experiments, might be expected to lead to non-specific effects, particularly after long time periods. It was not possible for us to carry out localisation studies after 16 h treatment with puromycin as both Ltk- and CHO cells appeared to be dying or dead after such treatment.
Stability of IRE-globin-myc transcripts
Using similar IRE-based constructs it has been found previously that degradation of chimeric globin-fos mRNA is linked to translation (15). Such a link for globin-myc would make it formally possible that the observed loss of localisation after desferrioxamine treatment was secondary to altered mRNA stability. The stability of the IRE-GM transcripts was estimated in actinomycin D chase experiments by measuring the abundance of globin transcripts over a 10 h period and calculating the time required for the mRNA abundance to fall by 50% (Table 1). The half-life of the GM transcripts was 7.9 h, which is considerably less than the half-life of globin mRNA (17-20 h) but much greater than that of c-myc alone (30 min). The half-life of the IRE-GM transcripts was also 7.9 h, showing that addition of the IRE alone did not affect stability of the transcript. Furthermore, after addition of iron to the medium or addition of the chelator desferrioxamine the half-life of IRE-GM transcripts was essentially unaffected, with estimated half-lives of 8.1 and 8.2 h, respectively. These data show that under conditions where globin-myc translation and localisation are altered there is no change in the mRNA stability. Thus, in the case of globin-myc transcripts, translation and stability are not linked and the observed reduced localisation when translation is reduced cannot be accounted for by a change in mRNA stability.
Table 1. Stability of chimeric globin-myc and IRE-globin-myc mRNAs
| Growth conditions | Cell line | Globin transcript half-life (h) |
| Normal DMEM | GM | 7.9 ± 0.4 |
| Normal DMEM | IRE-GM | 7.9 ± 0.7 |
| Ferric ammonium chloride (10 µM) | IRE-GM | 8.2 ± 0.3 |
| Desferrioxamine (100 µM) | IRE-GM | 8.1 ± 1.1 |
CONCLUSIONS
The 3[prime]-UTR is required for both the perinuclear localisation of c-myc mRNA and its association with the cytoskeleton (6); furthermore, it is sufficient to target a [beta]-globin reporter to these locations (7). The present data show that modulation of translation by regulation of a heterologous IRE can prevent localisation by the c-myc 3[prime]-UTR.
Intracellular iron regulates translation of ferritin mRNA by controlling protein binding to the IRE. When iron concentrations are high no protein binding occurs and translation proceeds, whereas when iron is depleted, as in the presence of desferroxiamine, the protein binds to the IRE and represses translation by reducing further binding of initiation factors to the mRNA and so preventing formation of a pre-initiation complex (14). The present results show that when iron was depleted over a 16 h period the IRE-GM transcripts were not localised, indicating that repression of IRE-GM translation by prevention of intiation complex formation reduced localisation. Thus, the data indicate that some form of initiation complex is required for the mRNA to be localised.
The implication of this is that the mRNA is localised bound to some or all of the protein components of the translation initiation complex. Such a view is compatible with the observations that myelin basic protein mRNA is transported in granules which contain other components of the protein synthetic apparatus including translation initiation factors (13). Further studies are required to identify the nature of the factors required for localisation and the precise nature of the RNA-protein complex that is localised. In contrast, once the mRNA has been localised continued translation is not necessary for localisation of transcripts targeted by the c-myc 3[prime]-UTR or [beta]-actin mRNA, as shown by the lack of effect of puromycin on localisation (Fig. 3; 16).
Although reduced translation prevented IRE-GM localisation it had no effect on stability of the transcripts. Thus, in the case of these transcripts degradation of the mRNA is not linked to mRNA translation, unlike the case of c-fos and chimeric globin-fos transcripts where mRNA degradation is dependent on translation (15). These data also imply that perinuclear localisation by the myc 3[prime]-UTR is independent of mRNA stability, giving support to earlier observations suggesting that localisation by the c-myc 3[prime]-UTR was not associated with mRNA instability (7). Indeed, the c-myc 3[prime]-UTR contains three regions conserved between species (19), of which the two which have been implicated in determination of mRNA stability are distinct from the one involved in mRNA localisation. The link between localisation by a 3[prime]-UTR signal and modulation of translation by a 5[prime]-UTR sequence may be pertinent to the on-going argument over whether interactions between 3[prime]- and 5[prime]-ends of mRNAs are important in post-transcriptional control and translation (20).
ACKNOWLEDGEMENTS
This work was supported by the Scottish Office Agriculture, Environment and Fisheries Department and by the European Community (BioMed BHM4 CT95-0995).
REFERENCES
*To whom correspondence should be addressed. Tel: +44 1224 716636; Fax: +44 1224 716622; Email: jeh{at}rri.sari.ac.uk
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