| Nucleic Acids Research | Pages |
A single nucleotide change in the c-myc internal ribosome entry segment leads to enhanced bindingof a group of protein factors
Introduction
Materials And Methods
Cell culture
Polymerase chain reaction
In vitro RNA synthesis
Cell extract (CE) preparation
Electrophoretic mobility shift assays (EMSAs)
North-western blotting
UV cross-linking assay
Results
Investigation of the affinity of cytoplasmic proteins for the Wt and Mt c-myc 5[prime] UTR RNA transcripts
Identification of specific c-myc 5[prime] UTR-protein complexes
North-western blotting
UV cross-linking analysis
UV competition assays
Discussion
Acknowledgements
References
A single nucleotide change in the c-myc internal ribosome entry segment leads to enhanced bindingof a group of protein factors
ABSTRACT
INTRODUCTION
The c-myc protein is a key regulator in the processes of cellular proliferation, growth cycle progression and differentiation (1). A variety of different growth stimuli induce c-myc expression within the first 2 h of G1 and this induction is sufficient to cause quiescent cells to enter S phase (2,3). In contrast, c-myc expression is down-regulated upon growth arrest and inhibition of c-myc expression using antisense oligonucleotides will prevent mitogen-treated cells from entering S phase (3). The c-myc protein functions as a transcription factor and along with its binding partner, Max, forms heterodimers which bind to a hexanucleotide sequence (CACGTG) known as an E box (4). Potential c-myc/Max target sequences include p53, eIF2[alpha], orinithine decarboxylase, eIF4E, CAD, uracil DNA glycosylase, [alpha] prothymosin and MrDb (5).
c-Myc protein levels are regulated by a wide range of different mechanisms. These include changes in the rate of transcription, occurring at both the initiation and elongation phases (6-8), alterations in the stability of the mRNA (9,10), changes in the half-life of the protein (11) and translational control mechanisms (12-16). In common with many other mRNAs encoding growth-related proteins, the 5[prime] untranslated region (UTR) of c-myc, which is encoded by exon 1 (3), is long and highly structured. Such structured 5[prime] UTRs can regulate translation by three major methods: (i) they can inhibit the normal scanning method of translation initiation [involving the binding of the eukaryotic initiation factor (eIF) eIF4E to the 7 methyl G cap, the helicase eIF4A and eIF4G (see 17 for review)]; (ii) they can contain upstream open reading frames (uORFs) which can cause translational inhibition (18); or (iii) they can contain internal ribosome entry segments (IRESs) and so direct cap-independent translation initiation (19-21). The c-myc 5[prime] UTR encoded by exon I was proposed originally by Saito et al. as a modulator of the translational efficiency (22) and, more recently, it has been shown that c-myc 5[prime] UTR contains an IRES which is capable of directing internal initiation of protein synthesis (15,16). The IRES lies 390 nucleotides upstream of the AUG translation start codon (16) thus meaning that c-myc transcripts which initiate from the major promoters P1 and P2, giving rise to 10-25 and 75-95% of cellular transcripts respectively (3), will contain the IRES. To date, very few eukaryotic IRESes have been identified and the situations where they are required remain elusive with c-myc providing the first example of a proto-oncogene which can be regulated in this manner (15,16). However, it should be noted that c-myc can additionally be translated in a cap-dependent manner since cells over-expressing the cap binding protein eIF4E, also display enhanced expression of c-myc (23).
Several forms of human neoplasia are associated with the overexpression of the endogenous c-myc gene. This can occur by gene amplification (24,25) and chromosomal translocations, e.g. in Burkitt's lymphoma involving the c-myc locus on human chromosome 8 to any one of the immunoglobulin loci on chromosome 2, 14 or 22 (26,27). Two situations of aberrant translational control of c-myc have also been described (28,29). In patients with multiple myeloma (MM), a disorder characterised by expansion of a plasma cell type in the bone marrow and osteolysis (30), a 20-fold increase in the amount of c-myc protein occurs by an aberrant translational mechanism (29). It appears that the increase in the c-myc protein levels in these cells is, in part, due to a 3.4-fold increase in the degree of association of the c-myc mRNA with the polysomes. This occurs without an increase in polysome size thus suggesting that there is an enhancement in the degree of mobilisation of this message from inactive mRNPs to polysomes rather than an alteration in the reinitiation rate (29). A consistent point mutation (C->T transition at 2756) was identified in all MM cell lines examined, which lies within the region subsequently shown by deletion analysis to contain the IRES (16).
In this work we have investigated the effect that this mutation has on the binding of protein factors to the c-myc 5[prime] UTR and show that there is enhanced binding of proteins to RNAs harbouring the mutation. In addition, we demonstrate that an MM-derived cell line expresses a different repertoire of proteins which bind to this region.
MATERIALS AND METHODS
Cell culture
The cell lines GM2132 (derived from an MM patient) and GM03201 (lymphoblastoid cell line derived from a healthy individual) were purchased from the American Type Culture Collection. Cells were grown in Roswell Park Memorial Institute medium (RPMI, GIBCO) containing 15% foetal calf serum in a 5% CO2 humidified atmosphere.
Polymerase chain reaction
Primers 5[prime]-GCCGGATCCCCGGCCCCTTTATAATGCGAG-3[prime] and 5[prime]-GTGGAATTCCTTTGGTTTTTTTCCCCAAACTACCCGAAA-3[prime], designed to contain restriction sites for BamHI and EcoRI, were synthesised and used to amplify c-myc exon 1 +/- the mutation (2289-2881; numbering as in 31). PCR reactions contained 10 µl PCR buffer (Advanced Biosystems), 10 µl MgCl2 (25 mM), 1 µl dNTPs (10 mM of each dATP, dCTP, dTTP, dGTP), 1 U of Taq DNA polymerase (Advanced Biosystems), 0.7 µg DNA and 20 µM of each oligonucleotide. The PCR reactions were carried out in a Perkin Elmer Cetus DNA thermal cycler at 94°C for 3 min followed by 37 cycles of (94°C for 2 min, 63°C for 3 min, 72°C for 2 min) and 72°C for 10 min. The resulting fragments (Fig.
Figure 1. The 600 bp section of the c-myc 5[prime] UTR was obtained by PCR. The position of the IRES and the mutation are shown. This fragment was subsequently subcloned into the vector Bluescript SK+.
In vitro RNA synthesis
Vectors were linearised downstream of inserts, phenol and chloroform extracted, and then ethanol precipitated. An aliquot of 1 µg of restricted template was incubated with 1 µl of each rNTP (10 mM) (rATP, rGTP, rCTP, rUTP), 1 µl 0.75 M DTT, 1 µl RNasin ribonuclease inhibitor (Promega), 5 µl 5× transcription buffer (Stratagene) and 10 U of T3 RNA polymerase at 37°C for 1 h. DNA templates were removed by addition of 10 U of RNase free DNase for 20 min at 37°C, and after phenol and chloroform extractions precipitated using 0.1 vol 3 M NaOAc (pH 5.2) and 2.5 vol ethanol. Radiolabelled transcripts were synthesised as above (for the UV cross-linking analysis additionally 1 mM 4-thio UTP was also included), and either 5 µl 800 Ci/mmol, 10 mCi/ml [[alpha]-32P]rCTP or rUTP in place of the appropriate unlabelled nucleotide. Unincorporated nucleotides were removed over a 1 ml Sephadex G-50 column. Transcript concentrations were determined by Cerenkov scintillation counting or by A260 values.
Cell extract (CE) preparation
Approximately 60-90 × 106 cells were harvested by centrifugation at 1000 r.p.m. for 5 min and washed in phosphate buffered saline. Cell pellets were resuspended in 600 µl lysis buffer, (0.5% NP-40, 300 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.4, 10% aprotinin, leupeptin and N-[vprop]-p-Tosyl-l-lysine chloromethyl ketone at 1 µg/ml and nuclei removed by subsequent centrifugation at 13 000 r.p.m. for 10 min.
Electrophoretic mobility shift assays (EMSAs)
Approximately 8.6 × 10-8 nmol labelled transcript (23 000 c.p.m.) were incubated in a 10 µl buffer mix containing 5 µl 5× transcription buffer (200 mM Tris-HCl pH 8.0, 40 mM MgCl2, 10 mM spermidine, 250 mM NaCl), 0.75 µl DTT (1 M), 1.5 µl tRNA (10 mg/ml), 1 µl rATP (10 mM), 1 µl glycerol and 0.17 µl vanadyl ribonucleoside complexes (10 mM). Nuclear free CEs were diluted in 0.01% DEPC-treated sterile, distilled water to a final volume of 20 µl and then incubated with the transcript mix at room temperature for 10 min. An aliquot of 3 µl 10× TBE loading buffer was added and samples loaded directly onto 0.7% agarose gels prepared using 1× TBE filter sterilised buffer. Samples were electrophoresed at 90 V for 2 h in 1× TBE filter sterilised buffer. Gels were dried under vacuum at 60°C for 2 h and exposed to Fuji-RX X-ray film at -70°C for 4-20 h.
North-western blotting
CEs were were separated by SDS-PAGE on either 7 or 10% gels. Proteins immobilised on nitrocellulose were allowed to renature by incubating the membranes in 5% BSA dissolved in 10-15 ml D67NP-40 solution [65% (v/v) D-Base (100 mM KCl, 0.2 mM K-EDTA, pH 8.0), 6.7 mM TEA pH 7.9, 170 mM DTT, 0.05% NP-40 filter sterilised] containing 1 mg/ml alkali denatured salmon testes DNA and 0.02 mg/ml yeast tRNA to block non-specific protein and nucleic acid binding sites. After at least 1 h incubation at room temperature, 32P-labelled RNA transcripts were added and then incubated for a further 1 h at 30°C. Membranes were washed in D67NP-40 solution and exposed directly to Fuji-RX X-ray film for 1-40 h at room temperature.
UV cross-linking assay
Radiolabelled RNA transcript (2.4 nmol; 4.5 × 105 c.p.m.) was incubated with 20 µg of CE in a 30 µl buffer mix (containing 10 mM HEPES pH 7.4, 3 mM MgCl2, 100 mM KCl, 1 mM DTT, 1 mM ATP, 6% glycerol) in the presence or absence of unlabelled competitor transcripts, for 10 min at room temperature in a 96-well microtitre plate (Falcon). The samples were then incubated for a further 10 min with heparin at a concentration of 0.2 mg/ml. Samples were UV-irradiated on ice for a period of 30 min using a 312 nm UV light source. RNase A (0.2 mg/ml) was added to each of the samples and incubated at 37°C for 30 min to allow degradation of any unprotected RNA species. An equal volume of 2× SDS sample buffer was added to the samples prior to separation by SDS-PAGE (7.5% gels). Gels were then stained using Coomassie blue, dried, and results visualised on a Molecular Dynamics PhosphorImager.
RESULTS
Investigation of the affinity of cytoplasmic proteins for the Wt and Mt c-myc 5[prime] UTR RNA transcripts
To analyse whether proteins were capable of binding to the c-myc 5[prime] UTR, EMSAs were performed whereby radiolabelled transcripts were incubated in the presence of cellular proteins. Nuclear free CEs were prepared from both an MM cell line, GM2132, and a control lymphoblastoid cell line, GM03201, which were found to be the most representative from each class as determined by FACS analysis (data not shown). The GM2132 cell line additionally has been shown to contain the highest level of c-myc protein of all the myeloma cell lines and hence could potentially contain enhanced levels of any putative trans-regulatory factors (29). Radiolabelled transcripts containing the Mt and Wt c-myc 5[prime] UTR (Fig.
Figure 2. EMSAs showing CE titrations using either MM (GM2132) or control (GM03201) CEs. Equivalent amounts of either Wt, Mt c-myc 5[prime] UTR RNA, transcripts were incubated with CEs as indicated. In each case, the addition of increasing amounts of CE resulted in the presence of less unbound transcript which was consequently associated with the elevated signal in higher molecular weight complexes. By comparing the electrophoretic mobility shift patterns obtained with the GM2132 CEs (MM) with those from the GM03201 CEs (control) it can be seen that the second shift is higher with the GM2132 CE than the GM03201 CE. This would tend to suggest that in the MM cell line there is an altered repertoire of RNA binding proteins. The electrophoretic mobility shift patterns obtained with the Wt and Mt c-myc 5[prime] UTR transcripts were very similar indicating that both RNA species probably bind the same proteins. However, for any given CE concentration, incubated with the same concentration of radiolabelled RNA, a higher proportion of the Mt transcript was present in a bound state compared to the equivalent Wt transcript. Thus, it appears that there is enhanced binding of proteins to the mutant transcript.
Identification of specific c-myc 5[prime] UTR-protein complexes
Two different techniques were employed in order to examine further the proteins which bind to the c-myc 5[prime] UTR: north-western blotting and UV cross-linking.
North-western blotting
Varying concentrations of nuclear free CEs from both the control (GM03201) and myeloma (GM2132) cell lines were size fractionated by SDS-PAGE and electroblotted on to nitrocellulose membranes. Proteins were allowed to partially renature on the membranes and then probed using radiolabelled transcripts. The results obtained using the Mt and Wt c-myc 5[prime] UTR, HRV-IRES and the GAPDH transcripts are shown (Fig.
Figure 3. North-western analysis of MM (GM2132) and control (GM03201) CEs. Different concentrations of CEs were separated by SDS-PAGE, blotted on to nitrocellulose and probed using equivalent amounts of radiolabelled RNA transcripts. (A) Wt c-myc 5[prime] UTR transcript. (B) Mt c-myc 5[prime] UTR transcript. (C) HRV-IRES transcript. (D) GAPDH transcript. Figure 4. North-western analysis of myeloma and control CEs in the presence of non-specific inhibitors. (A) Wt c-myc 5[prime] UTR transcript with 2 µg of unlabelled GAPDH transcript as non-specific competitor RNA. (B) Mt c-myc 5[prime] UTR transcript with 2 µg of unlabelled GAPDH transcript as non-specific competitor RNA. Proteins which are present at higher levels in MM extracts (GM2132) are marked (*). For each RNA transcript, a large number of RNA binding proteins were observed and although each one gave rise to a characteristic pattern of proteins, many were common to all of the transcripts. This suggests that they either represent non-specific RNA-binding proteins or bind RNA simply as a result of their high levels of expression. The latter is particularly evident at low protein concentrations. A number of differences were seen between the two cell lines; however, in general, the majority of these were not transcript specific implying that the myeloma cell lines have an altered spectrum of general RNA protein binding factors (Fig. The use of unlabelled GAPDH competitor (Fig. Predominantly, Wt c-myc 5[prime] UTR binding proteins, with molecular weights of 138, 82, 61 and 46 kDa were found in both GM2132 and GM03201 CEs, whilst proteins of 90 and 86 kDa appeared to be GM03201 CE and GM2132 CE specific respectively. Minimal binding to four other species of 79, 61, 76 and 70 kDa was also observed, the latter two of which were GM03201 CE specific. All of the aforementioned proteins were also found to bind the Mt transcript, albeit with higher affinity. This non-specific elevation in binding capacity of the Mt transcript was also accompanied by the binding of four additional proteins of 160, 98, 57 and 38 kDa which were not initially detected with the Wt transcript. In addition, there appears to be an increased level of proteins of 57 and 38 kDa in the myeloma-derived cell line. Longer exposures of the north-western probed with the Wt transcript (data not shown), in conjunction with the singular transcript north-western data, showed that the Wt transcript is also capable of binding these factors although with significantly lower affinity.
UV cross-linking analysis
UV cross-linking was performed to verify that the proteins identified by the previous method were indeed capable of directly interacting with the c-myc 5[prime] UTR RNA. Accordingly, [32P]CTP-radiolabelled Wt c-myc 5[prime] UTR transcripts were incubated with increasing amounts of GM2132 myeloma and GM03201 control CEs, proteins cross-linked to the RNA and unprotected RNA digested with RNase A. The proteins were then separated by SDS-PAGE and proteins binding RNA fragments visualised on a phosphorimager. Three major proteins were identified which interacted with the Wt c-myc 5[prime] UTR with molecular weights of ~105, 98 and 38 kDa (Fig.
Figure 5. UV cross-linking analysis of MM (GM2132) and control (GM03201) CEs. Different concentrations of CEs were incubated with radiolabelled c-myc Wt transcripts, exposed to UV light and then treated with RNase A (as described in Materials and Methods). A phosphorimage of the SDS-PAGE gel is shown. Proteins which are present at higher levels in MM extracts (GM2132) are marked (*).
UV competition assays
In order to investigate the specificity of the interactions of these proteins with the c-myc 5[prime] UTR, UV cross-linking analysis was performed using radiolabelled Wt transcripts in the presence of unlabelled Wt or Mt c-myc 5[prime] UTR, HRV-IRES and GAPDH transcripts. The amount of radioactivity in each RNA-protein complex was determined using a Molecular Dynamics PhosphorImager. In both cell lines, addition of an excess of unlabelled Wt/Mt c-myc 5[prime] UTR or HRV-IRES transcripts had an effect on the formation of the protein complexes when compared to GAPDH (Fig.
| Figure 6. UV cross-linking competition assays of protein complex-c-myc 5[prime] UTR with unlabelled RNAs. Twenty µg of MM (GM2132) (A) or control (GM03201) (B) CEs were incubated with the radiolabelled Wt c-myc 5[prime] UTR with increasing molar excesses of competitor RNAs as shown. A phosphorimage of the SDS-PAGE is shown. |
 |
The Mt transcript competed with the radiolabelled Wt RNA for binding to the 105 and 98 kDa proteins with 38 and 18% respectively of the radiolabelled protein complexes remaining after preincubation with a 30-fold molar excess and 11 and 9 % remaining after preincubation with a 100 molar excess (Fig.
DISCUSSION
We have described previously a C->T mutation at position 2756 (a region which we have subsequently shown to contain an IRES) in cell lines derived from patients with MM which show an increased expression of the c-myc protein by translational mechanisms (29). To investigate the effect of this mutation on protein binding and to determine whether the MM cells lines contain an altered spectrum of RNA binding proteins, three complementary approaches were employed.
The data obtained from the EMSAs demonstrated that the RNA sequences harbouring the mutation were able to enhance the binding of all proteins, a feature which we observed using all three techniques.
North-western blotting identified a wide range of general RNA binding proteins since the majority of these proteins were also found to bind the GAPDH transcript. However, addition of GAPDH as a competitor significantly reduced the non-specific binding and allowed the identification of putative c-myc 5[prime] UTR-specific binding proteins. Both the Wt and Mt c-myc 5[prime] UTR transcripts displayed similar protein binding potential, as predicted by virtue of the fact that only 1 nt out of 600 was altered. However, again there was enhancement in RNA binding affinity to the c-myc Mt transcript compared to the Wt sequence. For example, the Mt transcript bound more strongly to proteins of 98 and 38 kDa and, in addition, bound two other proteins of 160 and 57 kDa. Some differences in general RNA binding factors were also detected between the myeloma GM2132 and control GM03201 CEs (Table 1). This could account for the slight discrepancies in the electrophoretic mobility shift patterns obtained and may reflect the altered differentiated states of these B-cell clones. Alternatively, the additional proteins observed in the myeloma CEs may be attributable to the malignant phenotype of these cells.
Table 1.
| Method | GM2132 c-myc 5[prime] UTR-specific binding proteins (kDa) |
GM03201 c-myc 5[prime] UTR-specific binding proteins (kDa) |
Proteins which show enhanced binding to the mutant sequence (kDa) |
Summary of proteins binding to the c-myc5[prime] UTR (kDa) |
| North-western blotting | 86, 57, 38 | 90 | 160, 98, 57, 38 | 160, 138, 98, 61, 57, 38 |
| UV cross-linking | 86, 57, 38 | 105, 98 | 105, 98, 61, 57, 38 |
Proteins of 105, 98, 86, 61, 57 and 38 kDa were found to bind to both Mt and Wt-c-myc 5[prime] UTR transcripts in UV cross-linking experiments, although it could be seen that the myeloma cell lines had increased levels of the 86, 61, 55 and 38 kDa proteins (Table 1). Using excess unlabelled RNA transcripts we observed that the mutant sequences were able to compete more effectively than the Wt RNA for proteins with molecular weights of 105 and 98 kDa. Interestingly, proteins with molecular weights of 98 and 38 kDa have been shown previously to interact with the HRV-IRES and have been implicated in IRES function (32).
In conclusion, the north-western analysis and the UV cross-linking experiments imply that the C->T point mutation at 2756 is sufficient to enhance the interaction of a protein of 98 kDa to the c-myc 5[prime] UTR and our preliminary data suggest that this protein may bind around this 2756 site (F.E.M.Paulin and A.E.Willis, unpublished results). Several examples exist in viral systems which demonstrate that single mutations are capable of altering IRES function. For example, a single substitution in the FMDV-IRES has been shown to increase the degree of internal ribosome entry 1.5-5-fold (33). We speculate that this C->T mutation which we have found in cell lines derived from patients with MM combined with the altered protein repertoire could alter the initiation of translation through the IRES. However, the proof for this hypothesis awaits the purification of the factors involved and the investigation of their effects using functional assays.
ACKNOWLEDGEMENTS
This work was supported by project grants from the MRC (F.E.M.P.) and the Leukaemia Research Fund, grant 9654 (S.A.C).
REFERENCES
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