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. 2003 May 15;31(10):2508–2513. doi: 10.1093/nar/gkg357

Translational control of Scamper expression via a cell-specific internal ribosome entry site

Davide De Pietri Tonelli 1,2, Marija Mihailovich 1,2, Raphaela Schnurbus 1, Graziano Pesole 3, Fabio Grohovaz 1,2, Daniele Zacchetti 1,*
PMCID: PMC156039  PMID: 12736299

Abstract

The mRNA of Scamper, a putative intracellular calcium channel activated by sphingosylphosphocholine, contains a long 5′ transcript leader with several upstream AUGs. In this work we have investigated the role this sequence plays in the translational control of Scamper expression. The cytosolic transcription machinery of a T7 RNA polymerase recombinant vaccinia virus was used to avoid artifacts arising from cryptic promoters or mRNA processing. Based on transient transfection experiments of dicistronic and bi-monocistronic plasmids expressing reporter genes, we present evidence that the 5′ transcript leader of Scamper contains a functional internal ribosome entry site (IRES). Our data indicate that Scamper translation in Madin-Darby canine kidney cells is driven by a cap-independent mechanism supported by the IRES activity of its mRNA. Finally, the Scamper IRES appears to be the first IRES with specificity for kidney epithelial cells.

INTRODUCTION

It is generally considered that gene expression is mainly controlled at the transcriptional stage, and that the majority of mature transcripts are translated after cap-dependent ribosome recruitment, following the general rules of translation initiation (1). However, poor correlation between mRNA levels and protein expression (2) has recently drawn attention to the fundamental importance of post-transcriptional events in the regulation of protein expression. Among them are those involving mRNA processing, stability and localization as well as mRNA–protein interactions (3,4). Structural features of mRNA, such as stable secondary structures or upstream AUGs (1,5,6), may introduce further regulatory steps. Within this framework, the re-evaluation of the structure of the 5′ leaders of mature transcripts (7,8) has revealed unexpected complexity and potentially novel regulation of the translational process (9–11). In particular, recruitment of the small ribosomal subunit by an internal ribosome entry site (IRES) has been recognized as a mechanism of translation initiation alternative to cap-dependent ribosome recruitment (12,13). IRESes were originally discovered in some viruses, where they provide a way to bypass the shutdown of the host protein synthesis caused by the infection (14,15). Recently, several eukaryotic mRNAs have been proposed to contain IRES sequences as well (13,16). IRES elements do not share stringent sequence similarity, but bioinformatic analysis has predicted common features in their secondary structures (17). The list of cellular transcripts containing a putative IRES is growing fast and includes proto-oncogenes, growth factors, their receptors and homeodomain proteins (5,18). IRES elements are now envisaged as a way to preserve important physiological functions when cap-dependent translation is reduced, e.g. during mitosis (19,20) or cellular stress (21). Moreover, the control of protein expression by IRES elements can play an important role in development (18,22) and apoptosis (23). Recently, alteration of IRES function has been proposed to be involved in pathological conditions, such as Charcot-Marie-Tooth disease (24) and multiple myeloma (25).

IRESes are extensively used as biotechnological tools to express two genes in a single transcriptional unit (26). In fact, they are able to support the translation of a second cistron when inserted between two open reading frames in a dicistronic mRNA (27). This property is commonly exploited to demonstrate that the sequence under analysis contains an IRES, even though it has recently been pointed out that this is a necessary, but not sufficient, proof especially for IRESes with low activity (12).

In this work, we analyze the 5′ leader of Scamper mRNA, a cellular transcript isolated from Madin-Darby canine kidney (MDCK) cells (28), and originally suggested to encode for a receptor for sphingosylphosphocholine (29). By an approach based on the cytosolic transcription machinery of the vaccinia virus, we propose the presence of a functional IRES in the Scamper transcript. The specific activity of this novel IRES in kidney epithelial cells is discussed.

MATERIALS AND METHODS

Cell culture and transfection

Cells were maintained as follows: MDCK-II, MDBK, RK-13, Ptk-2 and A-72 in Earle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS) and 1 mM sodium pyruvate (A-72 only); SK-N-BE, HeLa and HEK-293T cells in Dulbecco’s modified Eagle’s medium containing 10% FCS, 1 mM sodium pyruvate (SK-N-BE only) and 1 mM non-essential amino acids (HEK-293T only); BHK-21 cells in Glasgow MEM with 5% FCS, 10% tryptose phosphate broth and 10 mM HEPES pH 7.2. All media were supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine, and cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

Cells were plated the day before the experiment in order to reach ∼70% confluence at the time of transfection. Cells were infected with the MVA-T7pol virus in MEM for 30 min at 37°C and then transfected for 6 h with plasmids carrying the DNA construct of interest. The transfection was performed with Superfect (Qiagen) according to the manufacturer’s instructions.

Cloning of plasmids

The cDNA of Renilla reniformis and Photinus pyralis (firefly) luciferases (from pRL-Tk and pSP-LUC+, respectively; Promega) were inserted into pBat4-mod (28) together with the EMCV IRES (from pIRES2-EGFP; Clontech) in the intercistronic region in order to obtain pBatmod2RL-IRES-LUC+ (D.Zacchetti, unpublished results). A synthetic double-stranded oligo with a T7 terminator and a T7 promoter (sense, TCG ATA ACC CCT TGG GGC CTC TAA ACG GGT CTT GAG GGG TTT TTT GCA GAT CTC GAG GCC TTA ATA CGA CTC ACT ATA G; antisense, TCG ACT ATA GTG AGT CGT ATT AAG GCC TCG AGA TCT GCA AAA AAC CCC TCA AGA CCC GTT TAG AGG CCC CAA GGG GTT A) was then inserted into pBatmod2-RL-IRES-LUC+ (using the SalI site between the Renilla luciferase and the EMCV IRES) to obtain the bi-monocistronic pBatmod2RL-monoIRES-LUC+. A portion of Scamper cDNA was amplified by PCR from total cDNA of MDCK cells (with primers TTA AGA TAC TTT TTC TAA AAA GAT TTA T and AGA GAG GTA CCT TTT AAG AGA GGA A) and the transcript leader inserted into pBatmod2-RL-IRES-LUC+ after removal of the EMCV IRES (pBatmod2-RL-ScaS6-LUC+) by blunting the NdeI site of the fragment and the NcoI site of the vector to reconstitute the start codon of the firefly luciferase. Shorter dicistronic constructs (pBatmod2-RL-ScaS4-LUC+, pBatmod2-RL-ScaS7-LUC+ and pBatmod2-RL-ScaS8-LUC+) and the control construct (pBatmod2-RL-ScaS6AS-LUC+) were also prepared by PCR in a similar way (details available on request). For expression driven by a nuclear promoter, the DNA encoding the dicistronic transcript was removed (AgeI–PstI) from either pBatmod2-RL-ScaS6-LUC+ or pBatmod2-RL-ScaS6AS-LUC+ and cloned into an AgeI/PstI-digested pEGFP-C3 plasmid (Clontech) to obtain pC3-RL-ScaS6-LUC+ and pC3-RL-ScaS6AS-LUC+, respectively. A promoterless plasmid was prepared by insertion of the ClaI–NsiI fragment from pBatmod2-RL-ScaS6-LUC+ into pZac (see below). Bi-monocistronic pBatmod2-RL-monoScaS6-LUC+ was constructed following the same strategy adopted for pBatmod2-RL-monoIRES-LUC+ starting from the corresponding dicistronic plasmid.

For in vitro translation the pZac plasmid was prepared by ligation of pBat4-mod (HindIII–PstI) with the following synthetic double-stranded oligos: thymidine kinase poly(A) (HindIII–XmaI), poly(A) (XmaI–PacI) and T7 terminator (PacI–PstI). Then, the SalI–NsiI fragment of pBATmod2-RL-ScaS6-Luc+ was inserted into SalI/NsiI-digested pZac to obtain pZac-ScaS6-Luc+.

In vitro translation

Firefly luciferase cRNA from the ApaI-linearized pZac-ScaS6-Luc+ plasmid was synthesized with the mMESSAGEmMACHINE kit (Ambion) according to the manufacturer’s instructions and purified with an RNeasy spin column (Qiagen). Cytosol was prepared from either MDCK or A-72 cells. Briefly, cells were detached with trypsin, washed with phosphate-buffered saline (PBS) and homogenized with a Dounce homogenizer in 1 vol of cold hypotonic MC buffer (10 mM HEPES-K pH 7.6, 10 mM K-acetate, 0.5 mM Mg-acetate, 5 mM DTT) supplemented with protease inhibitors (chymostatin, leupeptin, antipain and pepstatin A, each at 10 µg/ml). The homogenate was spun at 100 000 g and the supernatant (7.5 mg/ml protein) collected. In vitro translation reactions were performed with the ReticLysate IVT kit (Ambion) with 0.3 µg of cRNA and 4 µl of cytosol in a final volume of 25 µl according to the manufacturer’s instruction. Activity of the reporter gene was quantified with the Firefly luciferase assay system (Promega).

Luciferase reporter assay

Firefly and Renilla luciferase activities were revealed with the Dual-Luciferase reporter assay system (Promega) and measured (20 s readings) using a PlateLumino 96-well double injector luminometer (Stratec). Four independent reactions were carried out for each experimental point.

Bioinformatic tools

The IRES consensus structure in the transcript leader of Scamper mRNA has been detected by the UTRscan resource (30), designed to search user submitted sequences for UTR-specific functional patterns annotated in the UTRsite database (31).

RESULTS

Scamper transcript leader supports internal ribosome entry in MDCK and other kidney epithelial cells

In a previous work we re-evaluated the cDNA sequence of Scamper and showed that over-expression of this protein is toxic for cells (28). This observation brought us to search for regulatory mechanisms in Scamper expression. The discovery of a long (>350 bases) transcript leader with several upstream AUGs (Fig. 1A) led us to investigate the possible involvement of an IRES-driven cap-independent translation. In order to test this hypothesis, the transcript leader of Scamper was inserted in a dicistronic construct between two reporter genes and transiently transfected in MDCK cells, from which the Scamper cDNA was cloned. The activity of the 3′ cistron (firefly luciferase, Fluc) was normalized to the activity of the 5′ cistron (Renilla luciferase, Rluc), to account for variations in transfection efficiency. When transcription was driven by the cytomegalovirus promoter, an apparent IRES activity was detected (Fluc/Rluc ratio between 0.2 and 0.4). However, this result turned out to be affected by a cryptic promoter present in the intercistronic region. In fact, cloning the same dicistronic cassette into a promoterless vector revealed transcription and translation of the 3′ cistron (Fluc/Rluc ratio between 6 and 7.5). Nuclear DNA transfection of our constructs being open to artifacts, the expression of the reporter genes was driven by the MVA-T7pol vaccinia virus system. This strategy allows the transcription of the gene of interest to occur solely in the cytosol (32) thanks to the capping and polyadenylating activities of the viral enzymes (33). The dicistronic construct was cloned into a suitable vector (Fig. 1B) and transfected into either canine epithelial MDCK cells or canine fibroblast-like A-72 cells. Figure 1C shows the results obtained when Fluc translation was driven by either the 5′ leader of Scamper (Sca-S6) or the same sequence in the antisense orientation (Sca-S6as). Sca-S6 promoted a 4-fold increase in translation over Sca-S6as in MDCK cells (P < 0.005, paired two-sample Student’s t-test), but not in A-72 cells, where sense and antisense sequences showed comparable activity. This result rules out the possibility that a decrease in translation with the antisense construct is due to further inhibition of read-through and re-initiation. Moreover, use of Sca-S6as as the basal value of translation in dicistronic constructs is supported by the evidence that other luciferase-based dicistronic plasmids, characterized by long, highly structured and ATG-rich sequences in the intercistronic region, give comparable activity (D.De Pietri Tonelli and D.Zacchetti, unpublished observation). This body of evidence suggests that the translation observed in MDCK cells is due to the presence of an IRES. In agreement with this hypothesis, the translational activity was not inhibited when a stem–loop known to decrease the scanning of the ribosome (34) was inserted between the stop codon of the first cistron and the Sca-S6 sequence (data not shown).

Figure 1.

Figure 1

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The transcript leader of Scamper drives internal initiation of translation in MDCK but not A-72 cells. (A) The transcript leader of Scamper cDNA is represented with the upstream ATGs highlighted in bold and the start codon in upper-case letters. (B) Schematic representation of the dicistronic plasmid used for the expression of the reporter genes. (C) In MDCK cells, the efficiency of the dicistronic plasmid pBatmod2-RLScaS6-LUC+ (Sca-S6) containing Scamper transcript leader is ∼4-fold higher than pBatmod2-RL-ScaS6AS-LUC+ (Sca-S6as). In contrast, the two activities are comparable in canine A-72 cells. Ratios between signals from firefly (FLuc) and Renilla (RLuc) luciferases were measured as described in Materials and Methods. Results in this and the following figures are the means of at least four independent experiments.

By expressing the same amount of the two reporter genes in separate transcripts within the same cell type, we verified that the detection efficiency of Fluc is about one order of magnitude lower than Rluc in our experimental conditions (data not shown). According to this, the ratio value ∼0.02 in Figure 1 indicates that in vaccinia virus-infected MDCK cells, Sca-S6 driven translation is approximately five times less efficient than cap-dependent translation. Taking into account that the capping efficiency of the vaccinia virus machinery is ∼10% (33), the strength of the Scamper IRES is only one-fiftieth of the cap-dependent translation that can be obtained under the optimal context for translation initiation. This was also confirmed by a comparison with the well-known IRES from the encephalomyocarditis virus (EMCV). Sca-S6 efficiency was found to be two orders of magnitude lower when compared to the EMCV IRES in vaccinia virus-infected MDCK cells (data not shown).

In light of the different results obtained in MDCK and A-72 cells, we investigated the IRES activity of Sca-S6 in a larger panel of cells. Table 1 summarizes the results, expressed as the ratio between Sca-S6 and Sca-S6as translation values. Noticeably, Scamper IRES activity was significantly higher in epithelial cells from kidney than in other cell lines, thereby providing evidence in favor of a cell-specific control of translation.

Table 1. Scamper IRES activity in a panel of cell lines.

Cell line (species) Morphology, tissue ScaS6/ScaS6as (± SE)
MDCK (dog) Epithelial, kidney 4.0 (± 0.1)
RK-13 (rabbit) Epithelial, kidney 3.4 (± 0.3)
MDBK (cow) Epithelial, kidney 3.2 (± 0.2)
Ptk-2 (potoroo) Epithelial, kidney 3.0 (± 0.2)
SK-N-BE (human) Neuroblastoma 2.8 (± 0.5)
BHK (hamster) Fibroblast, kidney 2.5 (± 0.1)
A-72 (dog) Fibroblast, unknown tumor 1.5 (± 0.1)
HEK-293T (human) Fibroblastoid, kidney 1.5 (± 0.2)
HeLa (human) Epithelial, adenocarcinoma 1.3 (± 0.1)
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Ribosomal scanning through the Scamper transcript leader is inhibited in the presence of IRES activity

Since the dicistronic approach does not reflect a physiological context, we also evaluated whether cap-dependent ribosomal scanning contributes, together with IRES activity, to Scamper translation. We prepared a bi-monocistronic vector (m2Sca-S6) carrying the two reporter genes under separate transcriptional control of two T7 promoters. One reporter (Rluc) was placed in the same context for cap-dependent translation that was used in the dicistronic constructs, while the starting codon of the other (Fluc) was preceded by the 5′ leader of Scamper. In MDCK cells the translation efficiencies of Sca-S6 and m2Sca-S6 were comparable, thus indicating that translation is not further enhanced by cap-dependent ribosomal recruitment. However, this was not true for other cell lines where translation of Fluc in the bi-monocistronic plasmid largely exceeded that obtained in the corresponding dicistronic plasmid. Figure 2A shows the values of ratios between the activities of m2Sca-S6 and Sca-S6 in a panel of cell lines. Values around 1 indicate that only IRES activity accounts for translation in both conditions (dicistronic and bi- monocistronic plasmids), while higher values reveal a contribution of the cap-dependent scanning mechanism when the sequence is inserted in the bi-monocistronic vector. It can be noticed that when IRES activity is high, the cap-dependent scanning mechanism of translation is inhibited. The graph in Figure 2B highlights the correlation between these two parameters. We attempted to reproduce these results by a rabbit reticulocyte in vitro translation assay supplemented with cytosol from either MDCK or A-72 cells. Although the efficiency of the system did not allow the detection of the low Scamper IRES activity, a 50% inhibition of ribosomal scanning was clearly measurable when MDCK cytosol replaced A-72 cytosol in the in vitro translation reaction (M.Mihailovich and D.Zacchetti, unpublished observation).

Figure 2.

Figure 2

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Analysis of the translation efficiency driven by the Scamper transcript leader in various cell lines. (A) The ratio of the values of translation activity obtained with the bi-monocistronic and the dicistronic plasmids (m2Sca-S6/Sca-S6) is reported for a panel of cell lines. The corresponding IRES activities (as percentage of the effect obtained in MDCK cells) are included in parentheses. (B) A semi-logarithmic graph showing the correlation analysis of the values reported in (A), together with the correlation coefficient for a logarithmic fit.

Bioinformatic prediction of Scamper IRES structure

Finally, since the length of the Scamper 5′ leader can exceed that expected for a cellular IRES element (35), a series of 5′ deletion constructs were prepared, based on bioinformatic prediction, to determine whether the 5′ region of the transcript leader is essential for the IRES activity (Fig. 3A and B). In agreement with our expectations, Sca-S7 (98 nt in the intercistronic region) as well as Sca-S4 (a longer construct of 181 nt) maintained their ability to support internal initiation of translation (Fig. 3C), while the efficiency of a construct shorter than the predicted minimal domain (Sca-S8, 40 nt) was significantly lower. The residual translation activity of Sca-S8 was likely due to ribosomal read-through and re-initiation (27). As expected, neither Sca-S7 nor Sca-S4 was able to support internal initiation of translation in A-72 cells (data not shown).

Figure 3.

Figure 3

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IRES activity of 5′ deleted fragments of the Scamper transcript leader. A bioinformatic prediction of the secondary structure of the Scamper IRES (A) was used to design dicistronic vectors containing, in the intercistronic region, 5′ deleted fragments of the Scamper transcript leader (B). Among them, pBatmod2-RL-ScaS7-LUC+ (Sca-S7) is the one corresponding to the prediction. The activity of these dicistronic vectors was tested in MDCK cells (C). The translation efficiencies of pBatmod2-RL-ScaS6-LUC+ (Sca-S6), pBatmod2-RL-ScaS6AS-LUC+ (Sca-S6as), pBatmod2-RL-ScaS4-LUC+ (Sca-S4), pBatmod2-RL-ScaS7-LUC+ (Sca-S7) and pBatmod2-RL-ScaS8-LUC+ (Sca-S8) are shown along with the lengths of the corresponding intercistronic regions (insert length).

DISCUSSION

By a classical approach based on the expression of reporter genes in dicistronic constructs, we report evidence that the transcript leader of Scamper mRNA contains an IRES. The existence of an internal initiation of translation in cellular transcripts is appealing and supported by several reports. However, the validity of the dicistronic approach has been challenged by a recent review (12; see, however, 36). Taking into consideration the concerns in the definition of an internal initiation mechanism, we adopted stringent criteria. First of all, the antisense of the 5′ leader was employed to define a reference value of translation of the 3′ cistron. Although widely used, this strategy is open to criticisms, since the antisense sequence might depress translation, thus artificially enhancing the activity of the sense sequence. This possibility is unlikely in our conditions since other unrelated sequences, with similar length and ATG content, gave equivalent levels of background translation, and the activities of Sca-S6 and Sca-S6as in dicistronic constructs were comparable in some cell types, but not in others. Secondly, the reporter gene experiments were performed with the T7 RNA polymerase recombinant vaccinia virus, a system where transcription occurs exclusively at the cytosolic level. Since RNA analysis is considered of limited significance in the case of low IRES activity, because of possible nuclear processing of the transcript or presence of a cryptic promoter within the intercistronic region (12), the T7 RNA polymerase approach can be regarded as a way to avoid these two major sources of artifacts when defining an IRES.

The whole body of evidence we present here is fully consistent with the proposal that the Scamper transcript leader contains an IRES with an activity which is low when compared with the cap-dependent translation. Indeed, a moderate activity is expected considering the toxicity of Scamper under conditions of over-expression (28). However, under our experimental conditions, we cannot rule out the possibility that the lack of exposure to nuclear factors might affect the efficiency of translation, as proposed for other IRESes (37,38). Unfortunately, the presence of a cryptic promoter in the Scamper transcript leader makes testing this hypothesis problematic.

We also demonstrate that the Scamper IRES is able to drive internal initiation of translation in kidney epithelial cells, but not in other cell lines of various origins. This is not due to species specificity, but appears to be related to a tissue-specific mechanism of cap-independent translation. This result is in line with the recent proposal that the control of protein expression can occur not only at the transcriptional but also at the translational level with the participation of specific IRES elements (39,40). Interestingly, the comparison between dicistronic and monocistronic constructs in kidney epithelial cells reveals that a block of cap-dependent ribosomal scanning accompanies Scamper IRES activity. This implies that Scamper translation is virtually cap independent under physiological conditions, i.e. in monocistronic constructs. This block is not due to structural features of the transcript, since it is not observed in the cell lines where the IRES activity is low or negligible. Therefore, Scamper transcript leader per se can neither block ribosome scanning nor promote internal initiation of translation, supporting the idea that cell-specific trans-acting factors are required for both effects. Taking advantage of the tight correlation between IRES activity and ribosomal scanning block in various cell types, efforts will be devoted to the identification of the factors interacting with the transcript leader of Scamper.

Acknowledgments

ACKNOWLEDGEMENTS

We thank the other members of the Grohovaz laboratory for discussions and J. Meldolesi and D. Dunlap for reading the manuscript. We also thank G. Sutter and the Institute of Molecular Virology of the GSF–Forschungszentrum fuer Umwelt und Gesundheit GmbH for the MVA-T7pol virus. This work was carried out within the framework of the Italian Ministry of Research Center of Excellence in Physiopathology of Cell Differentiation. Financial support was also from the Armenise-Harvard Foundation, the Italian Telethon (grant E.0888 to F.G.), the CNR target project in Biotechnology (to F.G.) and the Ministero dell’Università e della Ricerca Scientifica (to G.P.) (‘Bioinformatics and Genomic Research, COFIN 99’, ‘Studio di geni di interesse biomedico e agroalimentare, legge 488/92’ and ‘Biotecnologie, legge 95/95’).

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