BACKGROUND
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  <dig> r gene, epb <dig>  is an example of a gene generating protein diversity.
it is best known for encoding protein  <dig> r, originally identified as an  <dig> kda component of the membrane skeleton of human red blood cells.
in these cells, protein  <dig> r stabilizes the spectrin-actin network and mediates its attachment to the overlying lipid bilayer through interactions with integral membrane proteins  <cit> .
while the expression pattern of  <dig> r in mature red cells is relatively simple, a rather complex array of  <dig> r protein isoforms of varying sizes  <cit>  and different subcellular locations  <cit>  has been reported in nucleated cells, indicating that protein  <dig> r plays roles at multiple sites in the cell.
the roles and partners of  <dig> r 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  <cit> .
protein  <dig> r is the founding member of a large family of proteins, the band  <dig>  superfamily, containing a highly conserved region designated 'the ferm domain'.
the domain takes its name from the  <dig>   and erm  proteins where it was discovered.
protein  <dig> r and three homologues of  <dig> r, namely  <dig> b ,  <dig> g  and  <dig> n , constitute the protein  <dig>  family.
ferm-containing proteins comprise a diverse group of eukaryotic proteins that bind membrane proteins and lipids and some of the members  are also involved in the organization of the actin cytoskeleton  <cit> .
the complex epb <dig> gene is approximately  <dig> kb long and is subject to extensive regulation at the level of alternative pre-mrna splicing  <cit> .
the regulated combinatorial use of at least  <dig> internal coding exons of the  <dig> r gene is responsible for the extensive range of  <dig> r isoforms, which differ with respect to their internal amino acid sequences.
additionally, two types of  <dig> r 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  translation-initiation codon responsible for the synthesis of the long isoforms of  <dig> r protein, .
short isoforms  are generated from mrnas from which exon 2' has been spliced out and translation is initiated at a downstream start site  present in exon  <dig>  <cit> .
recent studies have revealed great complexity in the 5' region of the  <dig> r 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  <dig> r isoforms in various cell types  <cit> .
a major challenge in the field is to explore the mechanisms by which a single  <dig> r 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  <cit> .
several less commonly used alternatives have also been identified that permit, in some cases, the production of isoforms differing in their n-terminal extensions  <cit> .
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  sequences.
ires elements were first discovered in  <dig> in rna of poliovirus and encephalomyocarditis virus  <cit> , 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  <cit> .
ires and ires trans-acting factors  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   <cit> .
in this study we show that  <dig> r mrnas containing the aug <dig> responsible for the synthesis of the long isoforms  have an ires element within the sequence located between the aug <dig> and the aug <dig> that allows the use of this internal site and hence the synthesis of the short   <dig> r isoforms.
the well-known itaf, ptb, binds to the ires-containing sequence.
our data reveal that:  aug1/aug2-containing mrnas can give rise to both long and short isoforms of protein  <dig> r;  the mechanism involved in the synthesis of the short isoforms is an internal entry of the ribosome;  the short isoforms of the  <dig> r protein can be generated from two different transcripts, those containing the aug <dig>  and those lacking the aug <dig> but containing the aug <dig>  <cit> .
these data suggest that the short  isoforms of protein  <dig> r are essential to the cell and that diverse mechanisms have therefore evolved to ensure their existence.
RESULTS
two proteins are synthesized in vitro from a specific set of  <dig> r cdnas
in this study we analyzed the in vitro expression of eleven  <dig> r cdnas, previously cloned by our group  <cit> , by coupled in vitro transcription and translation reactions using a t <dig> reticulocyte lysate system.
of the eleven  <dig> r cdnas, seven contained exon 2'  and, therefore, the upstream translation initiation site atg <dig>  which is used for the synthesis of long isoforms of protein  <dig> r .
the other four cdnas lacked exon 2'  and hence give rise to short isoforms of protein  <dig> r by using the downstream atg <dig> present in exon  <dig> as a translation initiation site .
for simplicity, we will name 1' to 7' the set of exon 2'-containing cdnas and  <dig> to  <dig> the set lacking exon 2'.
it should be mentioned that the long isoforms of protein  <dig> r are generally referred to as  <dig> r <dig> or ~ <dig> kda, although their sizes range from ~ <dig> kda to  <dig> kda.
similarly, the short isoforms of protein  <dig> r are generally referred to as  <dig> r <dig> or ~ <dig> kda, although their sizes range from ~ <dig> kda to  <dig> kda.
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  and, in addition, four of them produced a second product of faster electrophoretic mobility .
by analyzing the exon composition of cdnas 1' to 7', we noticed that those giving rise to two products all included exon  <dig> .
notably, cdnas 2' and 5' generated two and one  <dig> r product, respectively, and differed only by the presence  or absence  of exon  <dig> .
cdnas  <dig> to  <dig> use the atg <dig> present in exon  <dig> 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  <dig> to  <dig> was identical to that of the faster-migrating product synthesized from cdnas 1' to 4'.
these results suggest that the atg <dig> site is probably used in  <dig> r <dig> cdnas to synthesize the short  <dig> r products.
the short protein synthesized in vitro from  <dig> r <dig> cdnas is generated from the atg <dig> site
we next performed a directed mutagenesis experiment to replace the atg <dig> codon by gtg in two of the  <dig> r <dig> cdnas, 1' and 4' .
the mutant cdnas were in vitro-transcribed and translated, and the synthesized products analyzed by autoradiography .
the mutation completely abolished the synthesis of the short  <dig> r product from both  <dig> r <dig> cdnas, whereas the synthesis of the atg1-translated product remained unchanged .
these results are consistent with the hypothesis that the short protein originates from  <dig> r <dig> cdnas translated from the downstream atg <dig> site.
the short protein synthesized in vitro from  <dig> r <dig> cdnas is not produced by proteolysis
the results described above also suggest that the short  <dig> r product is unlikely to be produced from proteolytic cleavage of the long  <dig> r protein.
however, to rule out this possibility definitively we introduced a stop codon in cdna 4' upstream of the atg <dig> by site-directed mutagenesis .
figure 2b shows that, indeed, in the absence of the full-length atg1-translated product, the short protein product was still generated .
additionally, when the  <dig> r <dig> cdnas were expressed in vitro in the absence of the t <dig> promoter, no protein products were observed .
these results indicate that the  <dig> r 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  <dig> r <dig> cdnas containing exon 4
all previous experiments were developed in a coupled in vitro transcription-translation reticulocyte lysate system.
we next considered whether the atg <dig> site of  <dig> r <dig> cdnas was also used in living cells to give rise to the production of the short  <dig> r protein.
to investigate this, cdnas 1', 2', 3' and 4' were independently transfected in cos- <dig> cells and the expressed proteins were analyzed by western blot.
to detect the exogenous atg1-translated  <dig> r protein specifically we used the anti-flag antibody, since this epitope was added to the n-terminus of the long  <dig> r molecule.
to detect both the exogenous atg1- and atg2-translated  <dig> r proteins we used the anti-myc antibody as this epitope was added to the c-terminus of the  <dig> r molecule.
figure 3a and 3b shows representative results obtained for cdnas 1' and 4' that were similar to those observed for cdnas 2' and 3' .
as expected, the long protein was synthesized and recognized by both antibodies .
the short protein was also synthesized and detected by the anti-myc antibody but not by the anti-flag antibody .
the size of the short protein generated from cdnas 1' and 4' was similar to that of the atg2-translated  <dig> r isoform synthesized from the corresponding  <dig> r <dig> cdna .
these results indicate that the short protein most probably corresponds to the  <dig> r product synthesized from the atg <dig> site.
consistent with the results observed in vitro, all these data suggest that the atg <dig> site present in the set of  <dig> r <dig> cdnas containing exon  <dig> is indeed used in living cells.
generation of the short  <dig> r isoform is not due to a cryptic promoter
a cryptic promoter could be responsible for the production of mrna species giving rise to the short  <dig> r protein detected in the immunofluorescence and western blot analyses.
to test this possibility we eliminated the cytomegalovirus  promoter from the construct containing cdna 1'  and performed transfection experiments in cos- <dig> cells to analyze the protein expression pattern by western blot  using the anti-myc and the anti-flag antibodies.
the long and short  <dig> r protein bands were observed for the control cdna 1' construct, whereas removal of the cmv promoter resulted in a lack of synthesis of both  <dig> r proteins .
thus, the short  <dig> r protein detected in cos- <dig> cells transfected with the set of  <dig> r <dig> cdnas containing exon  <dig> is not due to the existence of an internal promoter.
the 5' sequence upstream of the atg <dig> site directs internal translation from a bicistronic vector
having ruled out the existence of a cryptic promoter and proteolytic cleavage as the mechanisms for generating the short  <dig> r isoforms, we considered internal translation to be the most plausible mechanism responsible for their synthesis.
to test this hypothesis, a sequence comprised of  <dig> nucleotides upstream of the atg <dig>  of  <dig> r was inserted into a bicistronic plasmid between the renilla and firefly luciferase reporter genes .
bicistronic plasmids containing the  <dig> nucleotides in an antisense orientation  or the foot-and-mouth disease virus  ires  inserted between the two cistrons were used as negative and positive controls, respectively .
these plasmids were independently transfected into cos- <dig> cells and the expressed proteins were detected by enzymatic activity assays.
as shown in figure 5b, cell lysates transfected with rluc- <dig> s-fluc were positive for firefly luciferase suggesting that the cloned  <dig> nucleotides of  <dig> r contain a functional ires.
expression of the second cistron directed by the  <dig> r sequence was lower than that directed by the fmdv ires.
a second bicistronic system consisting of dsred and enhanced green fluorescence protein  as first and second reporter cistrons, respectively, corroborated the results obtained with the renilla/firefly luciferase system .
as the fluorescence intensity of dsred and egfp is very easily measured by flow cytometry, we adopted this system for the rest of the experiments in this study.
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  following transfection of cos- <dig> cells with the bicistronic vector dsred- <dig> s-egfp or with dsred- <dig> a-egfp or with a plasmid coding only for egfp , which was used as monocistronic transcript control .
a band corresponding to the expected size of  <dig> 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 .
monocistronic egfp mrna  species were not detected in any of the bicistronic vectors used .
an ires element located in the region 5' upstream of the atg <dig> directs translation of the second cistron
a positive bicistronic test indicates that an alternate initiation mechanism is involved in the production of the short  <dig> r isoform.
a number of internal translational initiation mechanisms have been described: reinitiation, leaky scanning, shunting and internal ribosome entry  <cit> .
reinitiation cannot explain the generation of the short  <dig> r isoforms.
to distinguish between the other three possibilities, we generated an additional bicistronic construct in which a synthetic stem loop with a Δg° = - <dig> kcal/mol, which is known to decrease the scanning of the ribosome  <cit> , was inserted either 5' to the first cistron, hdsred- <dig> s-egfp, or between the stop codon of the first cistron and the  <dig> r sequence, dsredh- <dig> s-egfp .
these constructs and the original dsred- <dig> s-egfp construct were independently transfected in cos- <dig> cells and expression of dsred and egfp was determined by flow cytometry.
figure 6b illustrates that the hairpin abolished the expression of the first cistron when added to its 5' end but not the expression of the second cistron .
the second cistron was also expressed when the hairpin was added downstream of the dsred reporter gene .
these results indicate that the  <dig> r sequence comprised between the atg <dig> and the atg <dig> contains an internal ribosome entry site.
the length of the  <dig> r sequence between the two reporter genes determines the efficiency of the second cistron translation
to determine the influence of the 5' region upstream of the atg <dig> on the usage of the second cistron, we prepared three different 5' deletion constructs .
as negative controls, we used the same  <dig> r sequences in antisense orientations.
all these constructs were transfected in cos- <dig> cells and fluorescence intensity was analyzed by flow cytometry .
the construct lacking  <dig> nucleotides from the 5' region of exon  <dig>  had slightly lower efficiency than the construct with the entire sequence .
the other two deletion constructs had very low efficiencies, almost comparable with that of their corresponding antisense controls.
thus, removal of 5' sequences negatively affects the translation of the second cistron, being nucleotides  <dig> to  <dig> essential for ires activity.
the  <dig> r sequence containing the ires interacts with ptb
although the precise mechanism by which cellular iress promote translation is not fully understood, the participation of ires-binding proteins, referred to as itafs, is well documented  <cit> .
to analyze proteins interacting with the  <dig> nucleotide segment containing the  <dig> r ires, we performed uv-crosslinking experiments.
reticulocyte lysates were used as a source of itafs.
as a control we used a  <dig> r riboprobe lacking ires activity, the 149-ribonucleotide fragment corresponding to the 3' terminal end of the  <dig> ribonucleotide sequence .
figure  <dig> shows that the 32p-labelled  <dig> riboprobe crosslinked proteins ranging in size from  <dig> to  <dig> kda which, by contrast, the  <dig> ribonucleotide fragment did not crosslink.
the crosslinking of these proteins was globally competed by incubation with a 150-fold molar excess of the unlabelled  <dig> riboprobe.
a protein of approximately  <dig> kda was one of the most prominent bands bound to the  <dig> riboprobe.
as ptb is a ~ <dig> kda protein that interacts with pyrimidine-rich sequences of many viral and cellular iress, we analyzed the possible interaction of ptb with the  <dig> riboprobe.
figure 8b shows that the  <dig> but not the  <dig> riboprobe crosslinked purified, recombinant ptb.
this result is consistent with the presence of a polypyrimidine-rich tract in the large fragment, which is absent from the short one.
discussion
non-erythroid cells express a wide range of  <dig> r 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  <dig> r pre-mrna and the use of different translation-initiation sites  <cit> .
we report here an additional layer of complexity in the already complex  <dig> r 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  <dig> r. therefore, mammalian  <dig> r 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  <dig> r isoforms with different amino-terminal regions, long and short isoforms, were synthesized from two sets of mrnas generated by alternative splicing of the  <dig> r pre-mrna  <cit> .
the long isoforms are generally referred to as  <dig> r <dig> or  <dig> kda although their molecular masses vary from ~ <dig> to  <dig> kda.
the short isoforms are generally referred to as  <dig> r <dig> or  <dig> kda, although their molecular masses vary from ~ <dig> to  <dig> kda.
the two types of  <dig> r mrnas differ solely by the presence or absence of  <dig> nt, depending on whether exon 2' was maintained or skipped, respectively, implying that proteins  <dig> r <dig> are synthesized from mrnas containing a rather long 5' utr .
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  <cit> .
it has always intrigued us whether the long 5' utr in the  <dig> r mrnas responsible for the generation of  <dig> r <dig> 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  and, more importantly, the synthesis of the short isoforms of proteins  <dig> r, both in vivo and in vitro,  from  <dig> r mrnas containing exon 2' and most probably from  <dig> r mrnas skipping exon 2'.
a protein that is considered to be a general itaf, ptb  <cit> , is bound to the ires-containing region but not to an inactive fragment immediately adjacent to the aug <dig> .
our finding that a single  <dig> r cdna can give rise to two  <dig> r 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  <cit> .
interestingly, the ires element identified in  <dig> r is not only derived from within the coding region of the mrnas containing exon 2' but also in the 5' utr of  <dig> r 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  <cit> .
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  <dig> r, were examined in this study.
our data  indicated that the short isoforms of protein  <dig> r 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 .
rna cleavage and processing were also ruled out .
our data clearly imply that internal entry of the ribosome to the aug <dig> is responsible for the generation of the short isoforms of proteins  <dig> r.
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  <dig> r <dig> isoforms were synthesized from  <dig> r <dig> cdnas containing exon  <dig> in in vitro translation assays and in in vivo transfection experiments performed in non-synchronized cells.
consistently, no altered production of  <dig> r <dig> 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 .
these observations imply that the short isoforms of protein  <dig> r,  <dig> r <dig>  may be generated constitutively by internal translation from aug1/aug2-containing mrnas.
given that the short isoforms of protein  <dig> r can be synthesized from their own mrnas, those lacking exon 2'  and containing exon  <dig> , what would then be the significance of their generation also by ires-mediated translation from mrnas containing aug1?
it is reasonable to hypothesize that  <dig> r <dig> 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  <cit> .
we  <cit>  and others  <cit>  have shown that a third translation-initiation codon  present in exon  <dig> originates ~ <dig> kda isoforms of protein  <dig> r from  <dig> r cdnas lacking exons 2'  and  <dig> .
this mrna species contains a 5' utr that is too long  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  <dig> kda isoform of protein  <dig> r we have detected a  <dig> kda band  suggesting that, indeed,  <dig> r mrnas may contain more ires elements than that described in this study.
CONCLUSIONS
the capacity of the  <dig> r gene, epb <dig>  to encode and differentially express many  <dig> r isoforms by alternatively spliced transcripts differing on translation initiation sites and internal sequences, makes epb <dig> 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  <dig> r generation is also controlled at the translational level.
the capacity of a single mrna species to produce two  <dig> r proteins by using an internal ribosome entry site adds a new level of complexity to the already complex regulation of  <dig> r expression.
our study emphasizes the need to take into account this new level of regulation to achieve a better understanding of the biology of  <dig> r proteins and their homologs, a large group of protein isoforms involved in the organization of the actin and microtubule cytoskeletons.

 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 r gene, epb is an example of a gene generating protein diversity. it is best known for encoding protein r, originally identified as an kda component of the membrane skeleton of human red blood cells. in these cells, protein r 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 r in mature red cells is relatively simple, a rather complex array of r protein isoforms of varying sizes and different subcellular locations has been reported in nucleated cells, indicating that protein r plays roles at multiple sites in the cell. the roles and partners of r 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. protein r is the founding member of a large family of proteins, the band superfamily, containing a highly conserved region designated'the ferm domain'. the domain takes its name from the and erm proteins where it was discovered. protein r and three homologues of r, namely b, g and n, constitute the protein family. ferm-containing proteins comprise a diverse group of eukaryotic proteins that bind membrane proteins and lipids and some of the members are also involved in the organization of the actin cytoskeleton. the complex epb gene is approximately kb long and is subject to extensive regulation at the level of alternative pre-mrna splicing. the regulated combinatorial use of at least internal coding exons of the r gene is responsible for the extensive range of r isoforms, which differ with respect to their internal amino acid sequences. additionally, two types of r 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 translation-initiation codon responsible for the synthesis of the long isoforms of r protein,. short isoforms are generated from mrnas from which exon 2' has been spliced out and translation is initiated at a downstream start site present in exon. recent studies have revealed great complexity in the 5' region of the r 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 r isoforms in various cell types. a major challenge in the field is to explore the mechanisms by which a single r 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. 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 sequences. ires elements were first discovered in in rna of poliovirus and encephalomyocarditis virus, 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 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. in this study we show that r mrnas containing the aug responsible for the synthesis of the long isoforms have an ires element within the sequence located between the aug and the aug that allows the use of this internal site and hence the synthesis of the short r isoforms. the well-known itaf, ptb, binds to the ires-containing sequence. our data reveal that: aug1/aug2-containing mrnas can give rise to both long and short isoforms of protein r; the mechanism involved in the synthesis of the short isoforms is an internal entry of the ribosome; the short isoforms of the r protein can be generated from two different transcripts, those containing the aug and those lacking the aug but containing the aug. these data suggest that the short isoforms of protein r are essential to the cell and that diverse mechanisms have therefore evolved to ensure their existence.  two proteins are synthesized in vitro from a specific set of r cdnas in this study we analyzed the in vitro expression of eleven r cdnas, previously cloned by our group, by coupled in vitro transcription and translation reactions using a t reticulocyte lysate system. of the eleven r cdnas, seven contained exon 2' and, therefore, the upstream translation initiation site atg which is used for the synthesis of long isoforms of protein r. the other four cdnas lacked exon 2' and hence give rise to short isoforms of protein r by using the downstream atg present in exon as a translation initiation site. for simplicity, we will name 1' to 7' the set of exon 2'-containing cdnas and to the set lacking exon 2'. it should be mentioned that the long isoforms of protein r are generally referred to as r or ~ kda, although their sizes range from ~ kda to kda. similarly, the short isoforms of protein r are generally referred to as r or ~ kda, although their sizes range from ~ kda to kda. 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 and, in addition, four of them produced a second product of faster electrophoretic mobility. by analyzing the exon composition of cdnas 1' to 7', we noticed that those giving rise to two products all included exon. notably, cdnas 2' and 5' generated two and one r product, respectively, and differed only by the presence or absence of exon. cdnas to use the atg present in exon 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 to was identical to that of the faster-migrating product synthesized from cdnas 1' to 4'. these results suggest that the atg site is probably used in r cdnas to synthesize the short r products. the short protein synthesized in vitro from r cdnas is generated from the atg site we next performed a directed mutagenesis experiment to replace the atg codon by gtg in two of the r cdnas, 1' and 4'. the mutant cdnas were in vitro-transcribed and translated, and the synthesized products analyzed by autoradiography. the mutation completely abolished the synthesis of the short r product from both r cdnas, whereas the synthesis of the atg1-translated product remained unchanged. these results are consistent with the hypothesis that the short protein originates from r cdnas translated from the downstream atg site. the short protein synthesized in vitro from r cdnas is not produced by proteolysis the results described above also suggest that the short r product is unlikely to be produced from proteolytic cleavage of the long r protein. however, to rule out this possibility definitively we introduced a stop codon in cdna 4' upstream of the atg by site-directed mutagenesis. figure 2b shows that, indeed, in the absence of the full-length atg1-translated product, the short protein product was still generated. additionally, when the r cdnas were expressed in vitro in the absence of the t promoter, no protein products were observed. these results indicate that the r 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 r cdnas containing exon 4 all previous experiments were developed in a coupled in vitro transcription-translation reticulocyte lysate system. we next considered whether the atg site of r cdnas was also used in living cells to give rise to the production of the short r protein. to investigate this, cdnas 1', 2', 3' and 4' were independently transfected in cos- cells and the expressed proteins were analyzed by western blot. to detect the exogenous atg1-translated r protein specifically we used the anti-flag antibody, since this epitope was added to the n-terminus of the long r molecule. to detect both the exogenous atg1- and atg2-translated r proteins we used the anti-myc antibody as this epitope was added to the c-terminus of the r molecule. figure 3a and 3b shows representative results obtained for cdnas 1' and 4' that were similar to those observed for cdnas 2' and 3'. as expected, the long protein was synthesized and recognized by both antibodies. the short protein was also synthesized and detected by the anti-myc antibody but not by the anti-flag antibody. the size of the short protein generated from cdnas 1' and 4' was similar to that of the atg2-translated r isoform synthesized from the corresponding r cdna. these results indicate that the short protein most probably corresponds to the r product synthesized from the atg site. consistent with the results observed in vitro, all these data suggest that the atg site present in the set of r cdnas containing exon is indeed used in living cells. generation of the short r isoform is not due to a cryptic promoter a cryptic promoter could be responsible for the production of mrna species giving rise to the short r protein detected in the immunofluorescence and western blot analyses. to test this possibility we eliminated the cytomegalovirus promoter from the construct containing cdna 1' and performed transfection experiments in cos- cells to analyze the protein expression pattern by western blot using the anti-myc and the anti-flag antibodies. the long and short r protein bands were observed for the control cdna 1' construct, whereas removal of the cmv promoter resulted in a lack of synthesis of both r proteins. thus, the short r protein detected in cos- cells transfected with the set of r cdnas containing exon is not due to the existence of an internal promoter. the 5' sequence upstream of the atg site directs internal translation from a bicistronic vector having ruled out the existence of a cryptic promoter and proteolytic cleavage as the mechanisms for generating the short r isoforms, we considered internal translation to be the most plausible mechanism responsible for their synthesis. to test this hypothesis, a sequence comprised of nucleotides upstream of the atg of r was inserted into a bicistronic plasmid between the renilla and firefly luciferase reporter genes. bicistronic plasmids containing the nucleotides in an antisense orientation or the foot-and-mouth disease virus ires inserted between the two cistrons were used as negative and positive controls, respectively. these plasmids were independently transfected into cos- cells and the expressed proteins were detected by enzymatic activity assays. as shown in figure 5b, cell lysates transfected with rluc- s-fluc were positive for firefly luciferase suggesting that the cloned nucleotides of r contain a functional ires. expression of the second cistron directed by the r sequence was lower than that directed by the fmdv ires. a second bicistronic system consisting of dsred and enhanced green fluorescence protein as first and second reporter cistrons, respectively, corroborated the results obtained with the renilla/firefly luciferase system. as the fluorescence intensity of dsred and egfp is very easily measured by flow cytometry, we adopted this system for the rest of the experiments in this study. 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 following transfection of cos- cells with the bicistronic vector dsred- s-egfp or with dsred- a-egfp or with a plasmid coding only for egfp, which was used as monocistronic transcript control. a band corresponding to the expected size of 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. monocistronic egfp mrna species were not detected in any of the bicistronic vectors used. an ires element located in the region 5' upstream of the atg directs translation of the second cistron a positive bicistronic test indicates that an alternate initiation mechanism is involved in the production of the short r isoform. a number of internal translational initiation mechanisms have been described: reinitiation, leaky scanning, shunting and internal ribosome entry. reinitiation cannot explain the generation of the short r isoforms. to distinguish between the other three possibilities, we generated an additional bicistronic construct in which a synthetic stem loop with a Δg° = - kcal/mol, which is known to decrease the scanning of the ribosome, was inserted either 5' to the first cistron, hdsred- s-egfp, or between the stop codon of the first cistron and the r sequence, dsredh- s-egfp. these constructs and the original dsred- s-egfp construct were independently transfected in cos- cells and expression of dsred and egfp was determined by flow cytometry. figure 6b illustrates that the hairpin abolished the expression of the first cistron when added to its 5' end but not the expression of the second cistron. the second cistron was also expressed when the hairpin was added downstream of the dsred reporter gene. these results indicate that the r sequence comprised between the atg and the atg contains an internal ribosome entry site. the length of the r sequence between the two reporter genes determines the efficiency of the second cistron translation to determine the influence of the 5' region upstream of the atg on the usage of the second cistron, we prepared three different 5' deletion constructs. as negative controls, we used the same r sequences in antisense orientations. all these constructs were transfected in cos- cells and fluorescence intensity was analyzed by flow cytometry. the construct lacking nucleotides from the 5' region of exon had slightly lower efficiency than the construct with the entire sequence. the other two deletion constructs had very low efficiencies, almost comparable with that of their corresponding antisense controls. thus, removal of 5' sequences negatively affects the translation of the second cistron, being nucleotides to essential for ires activity. the r sequence containing the ires interacts with ptb although the precise mechanism by which cellular iress promote translation is not fully understood, the participation of ires-binding proteins, referred to as itafs, is well documented. to analyze proteins interacting with the nucleotide segment containing the r ires, we performed uv-crosslinking experiments. reticulocyte lysates were used as a source of itafs. as a control we used a r riboprobe lacking ires activity, the 149-ribonucleotide fragment corresponding to the 3' terminal end of the ribonucleotide sequence. figure shows that the 32p-labelled riboprobe crosslinked proteins ranging in size from to kda which, by contrast, the ribonucleotide fragment did not crosslink. the crosslinking of these proteins was globally competed by incubation with a 150-fold molar excess of the unlabelled riboprobe. a protein of approximately kda was one of the most prominent bands bound to the riboprobe. as ptb is a ~ kda protein that interacts with pyrimidine-rich sequences of many viral and cellular iress, we analyzed the possible interaction of ptb with the riboprobe. figure 8b shows that the but not the riboprobe crosslinked purified, recombinant ptb. this result is consistent with the presence of a polypyrimidine-rich tract in the large fragment, which is absent from the short one. discussion non-erythroid cells express a wide range of r 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 r pre-mrna and the use of different translation-initiation sites. we report here an additional layer of complexity in the already complex r 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 r. therefore, mammalian r 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 r isoforms with different amino-terminal regions, long and short isoforms, were synthesized from two sets of mrnas generated by alternative splicing of the r pre-mrna. the long isoforms are generally referred to as r or kda although their molecular masses vary from ~ to kda. the short isoforms are generally referred to as r or kda, although their molecular masses vary from ~ to kda. the two types of r mrnas differ solely by the presence or absence of nt, depending on whether exon 2' was maintained or skipped, respectively, implying that proteins r are synthesized from mrnas containing a rather long 5' utr. 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 r mrnas responsible for the generation of r 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 and, more importantly, the synthesis of the short isoforms of proteins r, both in vivo and in vitro, from r mrnas containing exon 2' and most probably from r 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 aug. our finding that a single r cdna can give rise to two r 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. interestingly, the ires element identified in r is not only derived from within the coding region of the mrnas containing exon 2' but also in the 5' utr of r 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 r, were examined in this study. our data indicated that the short isoforms of protein r 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. rna cleavage and processing were also ruled out. our data clearly imply that internal entry of the ribosome to the aug is responsible for the generation of the short isoforms of proteins r. 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 r isoforms were synthesized from r cdnas containing exon in in vitro translation assays and in in vivo transfection experiments performed in non-synchronized cells. consistently, no altered production of r 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. these observations imply that the short isoforms of protein r, r may be generated constitutively by internal translation from aug1/aug2-containing mrnas. given that the short isoforms of protein r can be synthesized from their own mrnas, those lacking exon 2' and containing exon, what would then be the significance of their generation also by ires-mediated translation from mrnas containing aug1? it is reasonable to hypothesize that r 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 present in exon originates ~ kda isoforms of protein r from r cdnas lacking exons 2' and. this mrna species contains a 5' utr that is too long 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 kda isoform of protein r we have detected a kda band suggesting that, indeed, r mrnas may contain more ires elements than that described in this study.  the capacity of the r gene, epb to encode and differentially express many r isoforms by alternatively spliced transcripts differing on translation initiation sites and internal sequences, makes epb 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 r generation is also controlled at the translational level. the capacity of a single mrna species to produce two r proteins by using an internal ribosome entry site adds a new level of complexity to the already complex regulation of r expression. our study emphasizes the need to take into account this new level of regulation to achieve a better understanding of the biology of r proteins and their homologs, a large group of protein isoforms involved in the organization of the actin and microtubule cytoskeletons. 
more importantly, similar results were also observed in cells transfected with this set of r cdnas. our study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of kda isoforms of protein r. this mechanism might also account for the generation of kda isoforms of r from a downstream aug our results reveal an additional level of control to r gene expression pathways and will contribute to the understanding of the biology of proteins r and their homologues, comprising an ample family of proteins involved in cytoskeletal organization. while the expression pattern of r in mature red cells is relatively simple, a rather complex array of r protein isoforms varying in n-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. by analyzing the in vitro expression of a panel of naturally occurring r cdnas, we observed that all aug1/aug2-containing cdnas gave rise to both long, kda, and short, kda, r isoforms. among these, kda and kda isoforms have different n-terminal extensions and are expressed either from aug1- or aug2-containing mrnas, respectively. mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from aug the presence of a cryptic promoter in the r cdna sequence was also discounted. in red blood cells, protein is an kda protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its ferm domain. when a nt sequence comprised between aug and aug was introduced into bicistronic vectors it directed protein expression from the second cistron.
 in red blood cells, protein is an kda protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its ferm domain. while the expression pattern of r in mature red cells is relatively simple, a rather complex array of r protein isoforms varying in n-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. among these, kda and 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 nucleotides which contain the aug codon, are produced by alternative splicing of the r pre-mrna. it is unknown whether the nt region comprised between aug and aug kept as a 5' untranslated region in aug2-containing mrnas, plays a role on r mrna translation.  by analyzing the in vitro expression of a panel of naturally occurring r cdnas, we observed that all aug1/aug2-containing cdnas gave rise to both long, kda, and short, kda, r isoforms. more importantly, similar results were also observed in cells transfected with this set of r cdnas. mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from aug the presence of a cryptic promoter in the r cdna sequence was also discounted. when a nt sequence comprised between aug and aug 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 nt sequence indicated that nucleotides to are essential for expression of the second cistron. the polypyrimidine tract-binding protein bound to the nt active sequence but not to an inactive 3'-fragment of nucleotides.  our study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of kda isoforms of protein r. this mechanism might also account for the generation of kda isoforms of r from a downstream aug our results reveal an additional level of control to r gene expression pathways and will contribute to the understanding of the biology of proteins r and their homologues, comprising an ample family of proteins involved in cytoskeletal organization. 