An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R
© Lospitao et al; licensee BioMed Central Ltd. 2008
Received: 08 August 2008
Accepted: 04 December 2008
Published: 04 December 2008
In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.
By analyzing the in vitro expression of a panel of naturally occurring 4.1R cDNAs, we observed that all AUG1/AUG2-containing cDNAs gave rise to both long, 135 kDa, and short, 80 kDa, 4.1R isoforms. More importantly, similar results were also observed in cells transfected with this set of 4.1R cDNAs. Mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from AUG2. The presence of a cryptic promoter in the 4.1R cDNA sequence was also discounted. When a 583 nt sequence comprised between AUG1 and AUG2 was introduced into bicistronic vectors it directed protein expression from the second cistron. This was also the case when ribosome scanning was abolished by introduction of a stable hairpin at the 5' region of the first cistron. Deletion analysis of the 583 nt sequence indicated that nucleotides 170 to 368 are essential for expression of the second cistron. The polypyrimidine tract-binding protein bound to the 583 nt active sequence but not to an inactive 3'-fragment of 149 nucleotides.
Our study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of 80 kDa isoforms of protein 4.1R. This mechanism might also account for the generation of 60 kDa isoforms of 4.1R from a downstream AUG3. Our results reveal an additional level of control to 4.1R gene expression pathways and will contribute to the understanding of the biology of proteins 4.1R and their homologues, comprising an ample family of proteins involved in cytoskeletal organization.
The mammalian proteome has been estimated to be at least an order of magnitude larger than its gene number. This highlights the importance of determining which genes give rise to protein diversity and the mechanisms involved in the generation of protein diversity from single genes.
The protein 4.1R gene, EPB41, is an example of a gene generating protein diversity. It is best known for encoding protein 4.1R, originally identified as an 80 kDa component of the membrane skeleton of human red blood cells. In these cells, protein 4.1R stabilizes the spectrin-actin network and mediates its attachment to the overlying lipid bilayer through interactions with integral membrane proteins . While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms of varying sizes [2, 3] and different subcellular locations [4–6] has been reported in nucleated cells, indicating that protein 4.1R plays roles at multiple sites in the cell. The roles and partners of 4.1R in non-erythroid cells are beginning to be elucidated. It turns out that they play structural roles, organizing membrane protein domains and/or linking membranes to internal cytoskeletal and nucleoskeletal networks [6–15]. Protein 4.1R is the founding member of a large family of proteins, the band 4.1 superfamily, containing a highly conserved region designated 'the FERM domain'. The domain takes its name from the 4.1 (four point one) and ERM (ezrin, radixin moesin) proteins where it was discovered. Protein 4.1R and three homologues of 4.1R, namely 4.1B (abundant in brain), 4.1G (general distribution) and 4.1N (abundant in neurons), constitute the protein 4.1 family. FERM-containing proteins comprise a diverse group of eukaryotic proteins that bind membrane proteins and lipids and some of the members (for instance, ERMs, talin, focal adhesion kinase, proteins 4.1) are also involved in the organization of the actin cytoskeleton .
The complex EPB41 gene is approximately 240 kb long and is subject to extensive regulation at the level of alternative pre-mRNA splicing [16–18]. The regulated combinatorial use of at least 10 internal coding exons of the 4.1R gene is responsible for the extensive range of 4.1R isoforms, which differ with respect to their internal amino acid sequences. Additionally, two types of 4.1R isoforms varying in their N-terminal extensions can be generated if exon 2' is maintained or spliced out. It is well known that inclusion of exon 2' retains the upstream AUG (AUG1) translation-initiation codon responsible for the synthesis of the long isoforms of 4.1R protein, (~135 kDa or 4.1R135). Short isoforms (~80 kDa or 4.1R80) are generated from mRNAs from which exon 2' has been spliced out and translation is initiated at a downstream start site (AUG2) present in exon 4 [19–21]. Recent studies have revealed great complexity in the 5' region of the 4.1R gene and have shown evidence of coupling between transcription and alternative splicing that directly affects alternative splicing of exon 2', thus regulating the synthesis of structurally different 4.1R isoforms in various cell types [22, 23]. A major challenge in the field is to explore the mechanisms by which a single 4.1R gene can express such a variety of isoforms with diverse structures and functions.
Regulation of translation plays a key role in gene expression control. Translation of eukaryotic mRNAs is mainly initiated by a linear scanning mechanism . Several less commonly used alternatives have also been identified that permit, in some cases, the production of isoforms differing in their N-terminal extensions [25–27]. Indeed, alternative translation initiation at different in-frame start codons is a process by which a single mRNA gives rise to proteins varying in their amino terminal extensions. Specific mRNAs bypass the linear ribosome scanning process by directly recruiting the ribosome and positioning it at the start codon through internal ribosome entry site (IRES) sequences. IRES elements were first discovered in 1988 in RNA of poliovirus and encephalomyocarditis virus [28, 29], two members of the Picornaviridae family. More recently, IRES activities have been detected in an increasing number of cellular mRNAs from yeast, Drosophila, birds and mammals, showing that the IRES process is far more extensive than previously thought. IRES-containing mRNAs encode a variety of proteins such as translation initiation factors, transcription factors, oncogenes, growth factors, homeotic genes and survival proteins . IRES and IRES trans-acting factors (ITAFs) collaborate in the recruitment of the 40S ribosomal subunit. It has been proposed that there are both specific ITAFs, which control the activity of related groups of IRESs, and general ITAFs (for example, polypyrimidine tract-binding protein, PTB) .
In this study we show that 4.1R mRNAs containing the AUG1 responsible for the synthesis of the long isoforms (~135 kDa) have an IRES element within the sequence located between the AUG1 and the AUG2 that allows the use of this internal site and hence the synthesis of the short (~80 kDa) 4.1R isoforms. The well-known ITAF, PTB, binds to the IRES-containing sequence. Our data reveal that: (i) AUG1/AUG2-containing mRNAs can give rise to both long and short isoforms of protein 4.1R; (ii) the mechanism involved in the synthesis of the short isoforms is an internal entry of the ribosome; (iii) the short isoforms of the 4.1R protein can be generated from two different transcripts, those containing the AUG1 (shown here) and those lacking the AUG1 but containing the AUG2 [19–21]. These data suggest that the short (~80 kDa) isoforms of protein 4.1R are essential to the cell and that diverse mechanisms have therefore evolved to ensure their existence.
Two proteins are synthesized in vitrofrom a specific set of 4.1R cDNAs
Focusing on the results of the in vitro expression of cDNAs 1' to 7', we observed that all of them gave rise to the expected ATG1-translated proteins (Figure 1D) and, in addition, four of them produced a second product of faster electrophoretic mobility (Figure 1D, lanes 1', 2', 3' and 4'). By analyzing the exon composition of cDNAs 1' to 7', we noticed that those giving rise to two products all included exon 4 (Figure 1B). Notably, cDNAs 2' and 5' generated two and one 4.1R product, respectively, and differed only by the presence (cDNA 2') or absence (cDNA 5') of exon 4 (Figure 1B). cDNAs 1 to 4 use the ATG2 present in exon 4 as a translation initiation site and their exon composition is similar to that of cDNAs 1' to 4', apart from lacking exon 2', so we compared the size of their in vitro-expressed products. Figure 1D shows that the size of the ATG2-translated products originating from cDNAs 1 to 4 was identical to that of the faster-migrating product synthesized from cDNAs 1' to 4'. These results suggest that the ATG2 site is probably used in 4.1R135 cDNAs to synthesize the short 4.1R products.
The short protein synthesized in vitro from 4.1R135cDNAs is generated from the ATG2 site
The short protein synthesized in vitro from 4.1R135cDNAs is not produced by proteolysis
The results described above also suggest that the short 4.1R product is unlikely to be produced from proteolytic cleavage of the long 4.1R protein. However, to rule out this possibility definitively we introduced a stop codon in cDNA 4' upstream of the ATG2 by site-directed mutagenesis (Figure 2A, 4'STOP). Figure 2B shows that, indeed, in the absence of the full-length ATG1-translated product, the short protein product was still generated (4'STOP). Additionally, when the 4.1R135 cDNAs were expressed in vitro in the absence of the T7 promoter, no protein products were observed (data not shown).
These results indicate that the 4.1R sequence does not contain a cryptic promoter directing the synthesis of the short protein product, and that the short product does not result from proteolytic cleavage of the long one.
In vivo expression of the set of 4.1R135cDNAs containing exon 4
Consistent with the results observed in vitro, all these data suggest that the ATG2 site present in the set of 4.1R135 cDNAs containing exon 4 is indeed used in living cells.
Generation of the short 4.1R isoform is not due to a cryptic promoter
The 5' sequence upstream of the ATG2 site directs internal translation from a bicistronic vector
The process directing translation of the second cistron in the bicistronic assay is not due to RNA processing or RNA cleavage
To investigate the possibility that RNA cleavage or RNA splicing contributed to the expression of the second reporter gene, we examined the integrity of the bicistronic transcript by northern blot analysis (Figure 5C) following transfection of COS-7 cells with the bicistronic vector DsRed-4.1s-EGFP or with DsRed-4.1a-EGFP or with a plasmid coding only for EGFP (pEGFP vector), which was used as monocistronic transcript control (Figure 5A). A band corresponding to the expected size of 2100 bp for the bicistronic RNAs was detected with an EGFP-specific cDNA probe from total RNA extracts isolated from cells transfected with the bicistronic vectors (Figure 5C). Monocistronic EGFP mRNA (720 bp) species were not detected in any of the bicistronic vectors used (compare lanes DsRed-4.1s-EGFP and DsRed-4.1a-EGFP with pEGFP).
An IRES element located in the region 5' upstream of the ATG2 directs translation of the second cistron
The length of the 4.1R sequence between the two reporter genes determines the efficiency of the second cistron translation
The 4.1R sequence containing the IRES interacts with PTB
Non-erythroid cells express a wide range of 4.1R isoforms that vary in N-terminal extensions and in internal sequences. This diversity is mainly generated by mechanisms coupling alternative promoter transcription, alternative splicing of the 4.1R pre-mRNA and the use of different translation-initiation sites [19–22]. We report here an additional layer of complexity in the already complex 4.1R gene regulation pathways based on our finding that alternative internal translation, more specifically an IRES-driven translation, is involved in the generation of the short isoforms of protein 4.1R. Therefore, mammalian 4.1R mRNA is added to the limited number of cellular polycistronic transcripts synthesizing two proteins harbouring different N-terminal regions by initiating translation from two in-frame AUG codons.
In the past, we learnt that two types of 4.1R isoforms with different amino-terminal regions, long and short isoforms, were synthesized from two sets of mRNAs generated by alternative splicing of the 4.1R pre-mRNA [19–21]. The long isoforms are generally referred to as 4.1R135 or 135 kDa although their molecular masses vary from ~97 to 135 kDa. The short isoforms are generally referred to as 4.1R80 or 80 kDa, although their molecular masses vary from ~62 to 80 kDa. The two types of 4.1R mRNAs differ solely by the presence or absence of 17 nt, depending on whether exon 2' was maintained or skipped, respectively, implying that proteins 4.1R80 are synthesized from mRNAs containing a rather long 5' UTR (more than 600 nt). Typically, mRNAs that are translated by the linear ribosome scanning mechanism contain short 5' UTRs; by contrast, mRNAs containing IRES elements keep long 5' UTRs . It has always intrigued us whether the long 5' UTR in the 4.1R mRNAs responsible for the generation of 4.1R80 proteins was maintained because it contained a relevant structured element. The present study demonstrates the existence of an IRES element within this region. The IRES element directs the synthesis of the second cistron in two bicistronic systems (Figure 5) and, more importantly, the synthesis of the short isoforms of proteins 4.1R, both in vivo and in vitro, (Figures 3 and 1, respectively) from 4.1R mRNAs containing exon 2' and most probably from 4.1R mRNAs skipping exon 2'. A protein that is considered to be a general ITAF, PTB , is bound to the IRES-containing region but not to an inactive fragment immediately adjacent to the AUG2 (Figure 8). Our finding that a single 4.1R cDNA can give rise to two 4.1R isoforms, long and short, establishes a new level of complexity that needs to be taken into account in future studies.
Most cellular IRES elements characterized to date are located in 5' UTR and few of them are located in coding regions [36–38]. Interestingly, the IRES element identified in 4.1R is not only derived from within the coding region of the mRNAs containing exon 2' but also in the 5' UTR of 4.1R mRNAs lacking exon 2'.
Some earlier studies analyzing cellular IRES elements did not rule out all the possible mechanisms involved in the generation of a shorter protein and, in some cases, the putative IRES sequence happened to be a cryptic promoter . It should be mentioned that mechanisms responsible for the generation of two isoforms from a single cDNA, such as transcription from a cryptic promoter or proteolytic cleavage from the large protein product of 4.1R, were examined in this study. Our data (Figures 4 and 2, respectively) indicated that the short isoforms of protein 4.1R were not generated by either of these two mechanisms. Similarly, the possible contribution of internal translation mechanisms such as shunting or leaky scanning to the internal initiation of translation was also examined in the bicistronic assay. A hairpin sequence capable of abolishing ribosome scanning was introduced at the 5' region of the first cistron and resulted in the failure to synthesize the first, but not the second, cistron (Figure 6). RNA cleavage and processing were also ruled out (Figure 5C). Our data clearly imply that internal entry of the ribosome to the AUG2 is responsible for the generation of the short isoforms of proteins 4.1R.
Many cellular IRES are relatively inactive in in vitro translation systems and, in cell assays, they are activated when cells are submitted to stress conditions or at some stages of the cell cycle. It is of note that large amounts of 4.1R80 isoforms were synthesized from 4.1R135 cDNAs containing exon 4 in in vitro translation assays and in in vivo transfection experiments performed in non-synchronized cells. Consistently, no altered production of 4.1R80 isoforms was detected when transfected cells were arrested in mitosis with nocodazole and then released by nocodazole wash-out or subjected to etoposide-induced apoptosis, serum starvation or heat-shock treatments (data not shown). These observations imply that the short isoforms of protein 4.1R, 4.1R80, may be generated constitutively by internal translation from AUG1/AUG2-containing mRNAs.
Given that the short isoforms of protein 4.1R can be synthesized from their own mRNAs, those lacking exon 2' (AUG1) and containing exon 4 (AUG2), what would then be the significance of their generation also by IRES-mediated translation from mRNAs containing AUG1? It is reasonable to hypothesize that 4.1R80 isoforms must be vital to the cell and that diverse mechanisms have therefore evolved to ensure their existence.
Examples of cellular proteins containing two separate IRES sequences have been described . We  and others  have shown that a third translation-initiation codon (AUG3) present in exon 8 originates ~60 kDa isoforms of protein 4.1R from 4.1R cDNAs lacking exons 2' (AUG1) and 4 (AUG2). This mRNA species contains a 5' UTR that is too long (more than 900 nt) for a linear ribosome scanning mechanism, which may be compatible with the existence of an IRES element. In some experiments, in addition to the generation of the 135 kDa isoform of protein 4.1R we have detected a 60 kDa band (data not shown) suggesting that, indeed, 4.1R mRNAs may contain more IRES elements than that described in this study.
The capacity of the 4.1R gene, EPB41, to encode and differentially express many 4.1R isoforms by alternatively spliced transcripts differing on translation initiation sites and internal sequences, makes EPB41 a paradigm of cellular economy in storing information. Our results show for the first time that, in addition to the regulation at the transcriptional and splicing levels, protein 4.1R generation is also controlled at the translational level. The capacity of a single mRNA species to produce two 4.1R proteins by using an internal ribosome entry site adds a new level of complexity to the already complex regulation of 4.1R expression. Our study emphasizes the need to take into account this new level of regulation to achieve a better understanding of the biology of 4.1R proteins and their homologs, a large group of protein isoforms involved in the organization of the actin and microtubule cytoskeletons.
Cell culture and transfection
COS-7 cells were grown as described . Transfection experiments were performed by electroporation using the Electro Cell Manipulator 600 (BTX, San Diego, CA). Cells were processed 48 hours after transfection.
Anti-FLAG antibody is a rabbit polyclonal antibody (Sigma, Saint Louis, MO). Anti-c-myc monoclonal antibody 9E10 was obtained from the American Type Culture Collection. Anti-horseradish peroxidase-labelled secondary antibodies were obtained from Southern Biotechnology Associates, Inc. Goat anti-rabbit immunoglobulin G (IgG) (H+L) secondary antibody conjugated to Alexa Fluor 488 was obtained from Molecular Probes.
4.1R cDNAs, 4.1R mutant constructs and in vitroprotein expression
4.1R cDNAs used for in vitro and in vivo expression were generated by RT-PCR using total RNA from MOLT-4 T cells as described previously [32, 33]. These cDNAs encoded 4.1R proteins tagged at the C-terminus with the c-myc epitope. Some of the cDNAs encoded 4.1R proteins also tagged at the N-terminus with the FLAG epitope. The cDNAs were cloned into pCR3.1 (Invitrogen) and verified by sequencing .
In two 4.1R cDNAs, previously designated 4.1R135Δ16 and 4.1R135Δ16,18,19 (for simplicity named in this study cDNAs 2' and 4', respectively) the ATG2 present in exon 4 was mutated and the adenine was substituted by guanosine, thus generating 1' ATG2mut and 4' ATG2mut, respectively. The mutant 4' STOP carried a nucleotide substitution at position 182 of exon 2 that changes the triplet AAG to the stop triplet TAG. These three mutant constructs were obtained using the QuikChange site-directed mutagenesis following the manufacturer's instructions (Stratagene). In vitro protein expression was achieved by coupled in vitro transcription and translation reactions, as previously described .
The DsRed coding sequence was amplified by PCR from the pDsRed1-N1 vector (Invitrogen) with the sense primer NheI-red (5'-CCGGTCGCTAGCATGGTGCGCTCC-3') and the antisense primer BglII-stop-red (5'-CTAGAGTAGATCTCGCTACAGGAA-3'). The PCR product was cloned between the NheI and BglII sites of pEGFP-N1 (Invitrogen).
Different 4.1R sequences, amplified by PCR, were inserted into the SacI site of the bicistronic vector. The sense primers used for the DsRed-4.1s-EGFP, DsRed-413s-EGFP, DsRed-215s-EGFP, DsRed-149s-EGFP construct were 5'-GGCCGGAGCTCCACAGCACCAAC-3', 5'-GACTTGACCGAGCTCAAGGAGCGGACA-3', 5'-CCCAATTGCAGAGCTCGAACCGGAAC-3' and 5'-GCAGAAACAGAGCTCGCTCAGGAAGAAC-3', respectively, and the antisense primer was 5'-GCAGTGCATGAGCTCGTGTTTTCTGATTGG-3' in all cases.
The hairpin structure (CCGGATCGG)3 described by Koromilas et al  was cloned between the XhoI and NheI sites of DsRed-4.1s-EGFP to generate the DsRedH-4.1s-EGFP and the HDsRed-4.1s-EGFP constructs, respectively.
The 4.1R sequence amplified with the 5'-GGCCGGAGCTCCACAGCACCAAC-3' and 5'-GCAGTGCATGAGCTCGTGTTTTCTGATTGG-3' primers was cloned into the SacI site of the RLuc-FLuc bicistronic vector in sense (RLuc-4.1s-FLuc) and antisense (RLuc-4.1a-FLuc) orientations.
Relative IRES activity was quantified in vivo as the ratio of the expression of firefly luciferase to that of Renilla luciferase in COS-7 cells transfected with the bicistronic constructs. Luciferase expression was determined from 1–2 × 105 cells using the dual luciferase reporter assay system following the manufacturer's instructions (Promega) in a luminometer (Berthold). Five independent experiments were performed in triplicate.
Northern blot analysis
Total RNA from COS-7 cells transfected with different constructs was extracted using TRIZOL Reagent (Invitrogen Life Technologies). For northern blot analysis, 20 μg of RNA were denatured in 50% formamide and 2.2 M formaldehyde at 65°C, subjected to electrophoresis in a 1% agarose/formaldehyde gel, and transferred to nylon membranes. RNA samples were hybridized under standard conditions to labelled EGFP cDNA. Final blot washing conditions were 0.5 × SSC/0.1% SDS (1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at 65°C.
To generate RNA riboprobes, PCR was performed with specific primers for the indicated 4.1R fragments to which additional sequences were added for incorporating the T7 RNA polymerase promoter at the 5' end. Radiolabeled RNA probes were prepared by transcription with T7 RNA polymerase in the presence of 0.08 mM unlabelled rUTP plus 25 μCi of (α-32P)UTP (400 Ci/mmol)(Amersham).
UV cross-linking assays
12.5 μl of rabbit reticulocyte lysates were incubated with radiolabeled probes at 30°C for 30 minutes. The reaction mixtures were exposed to UV (254 nm) (Stratalinker 1800; Stratagene) for 10 minutes on ice. Then 20 units of RNase A was added to the reaction and incubated during 10 minutes at 37°C. For competition experiments, a 150-molar excess of unlabelled RNA was added 10 minutes before the addition of the radiolabeled probe. For PTB-4.1R interaction experiments, 100 ng of recombinant His-PTB (a gift from Dr. J.M. Izquierdo, Centro de Biología Molecular Severo Ochoa, Madrid) was incubated with the appropriate radiolabeled probes. The RNA-protein complexes were resolved by SDS-PAGE.
COS-7 cells were fixed with 4% formalin (37% formaldehyde solution; Sigma), permeabilized, blocked, incubated with the appropriate antibodies, and processed as described . Controls with primary antibodies omitted were included in each experiment. Preparations were examined under a Zeiss epifluorescence microscope.
Western blot analysis
Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon polyvinylidine difluoride (Millipore) in Tris (tris(hydroxyl-methyl)aminomethane)-borate buffer, pH 8.2. Membranes were processed and developed as described .
Flow cytometry analysis
Transfected cells were detached from the dish and suspended at 0.5–1 × 106 cells/ml in phosphate-buffered saline, 2 mM EDTA. Samples were analyzed by flow cytometry using an argon laser at 488 and 558 nm to detect EGFP and DsRed expression, respectively, in a Calibur cytometer (Becton-Dickinson). Four to five independent experiments were performed in triplicate.
enhanced green fluorescence protein
four point one, ezrin, radixin and moesin
foot-and-mouth disease virus
internal ribosome entry site
IRES trans-acting factor
polypyrimidine tract-binding protein
The authors wish to thank Drs E Martínez-Salas, I Ventoso and JM Izquierdo (Centro de Biología Molecular Severo Ochoa, CBMSO, Madrid) for very valuable discussions and materials. We thank O Antón (CBMSO) for help with the Northern blot analysis. We also acknowledge A Pérez-González and S López de Quinto (CBMSO) for their initial input to this study. This work was supported by grants BFU2005-01825 and BFU2008-02460 from the Ministerio de Educación y Ciencia, and S-GEN-0166/2006 from the Comunidad de Madrid. EL was a postdoctoral fellow from the Comunidad de Madrid.
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