BACKGROUND
expression vectors containing an internal ribosome entry site  element have been widely used as bicistronic vectors that provide co-expression of two unrelated reading frames from a single transcript unit  <cit> .
a reading frame in a multiple cloning site downstream of a promoter is called the first cistron, and the second cistron is downstream of an ires element.
pireshyg <dig> is a bicistronic expression vector that possesses an intervening sequence  between the first cistron and an ires element derived from encephalomyocarditis virus, and a hygromycin resistance gene in the second cistron, which serves as a selection marker for stable transfection.
it has been shown that the first cistron gene is expressed at levels comparable to those achieved in a monocistronic vector and initiation of translation is cap-dependent  <cit> .
however, the present study is the first to show that the use of pireshyg <dig> affects the mrna stability of their carrying genes in mammalian cells, potentially leading to their insufficient expression.
nonsense-mediated mrna decay  is a post-transcriptional mrna quality control system that eliminates aberrant mrnas harboring premature termination codons  within protein coding regions in eukaryotes  <cit>  to protect the cells from accumulation of harmful or nonfunctional c-terminally truncated polypeptides  <cit> .
the degradation occurs in a translation-dependent manner when translation is initiated in an mrna cap-dependent manner  <cit> .
in mammalian cells, two determinants have been identified that distinguish "premature" termination codons from "normal" termination codons and provide a protective advantage to the normal termination codon  <cit> .
one is the presence of an exon-junction complex  more than  <dig> nucleotides downstream of a termination codon  <cit> .
induction of nmd requires the association between the ejc and the protein complex bound to the ribosome stalled at a ptc, which contains essential proteins to trigger nmd such as upf <dig>  eukaryotic release factors, and smg <dig>  <cit> .
because normal termination codons generally reside either in the final exon or within  <dig> nucleotides upstream of the 3'-end in the penultimate exon, the transcripts coding wild-type proteins are able to escape nmd  <cit> .
another determinant is the distance between the stop codon and a poly region  <cit> .
normal termination requires the interaction between the terminating ribosomal complex and the poly-binding proteins , which leads to faster release of a terminating ribosome from mrna  <cit> .
a ribosomal complex at a ptc fails to interact with pabps because of the relatively longer distance from the poly region, resulting in prolonged association with mrna, which stimulates nmd  <cit> .
recently, it has been reported that the length between a termination codon and poly region affects nmd efficiency, showing that longer 3'utr induces greater nmd activity  <cit> .
recently, we identified a novel splice variant of the granulocyte-macrophage colony-stimulating factor receptor  β chain  in patients with myelodysplastic syndrome, a clonal hematopoietic disorder  <cit> .
the splice variant  retained the 79-base intron v, resulting in a frameshift that introduced eighteen ptcs downstream of the retained intron.
when the cdnas encoding the βc <dig> or wild-type βc were cloned into the first cistron of pireshyg <dig> vector and stably expressed in a murine hematopoietic cell line, ba/f <dig>  βc <dig> mrna levels were significantly reduced compared with those observed for wild-type βc.
we show here that the pireshyg <dig> vector sensitizes the first cistron genes to nmd and, interestingly, that βc <dig> with ptcs was far more sensitive to the vector-dependent nmd resulting in minimized expression levels of the ptc-harboring gene.
RESULTS
intron v is not spliced out in βc <dig> clones
to determine whether the intron v was spliced in the cells stably expressing βc <dig>  we analyzed the presence of intron v in the integrated dna and respective rna isolated from each clone by pcr.
the locations of the sequences corresponding to the primers are shown in figure  <dig> and table  <dig>  pcr with 2s/2a primers that flank intron v distinguished βc <dig> cdna from wild-type βc cdna by the larger fragment size  .
in rna from all βc <dig> clones, only the larger size of pcr products was detectable.
the absence of a 387-bp band suggested that intron v in βc <dig> mrna was not spliced out in ba/f <dig> transfectants resulting in the generation of  <dig> ptcs.
the unspliced intron v in βc <dig> mrna was also detected by pcr using a 3s primer that was complementary to the 5'-end of intron v. all rna samples without reverse transcription failed to generate any pcr products .
*, the nucleotide position in the intron v-derived sequence
βc <dig> mrna decays faster than wild-type βc mrna
we determined the amounts of integrated dna and respective transcripts for wild-type βc or βc <dig> by real-time pcr with 1s/1a primers.
compared with a clone expressing wild-type βc 5c, βc <dig> clones me and m <dig> had similar amounts of integrated βc <dig> cdna, and about half of that amount was found in clones m <dig> and 63m .
however, as shown in figure 2b, in all four βc <dig> clones, rna levels for βc <dig> were less than 10% of that measured for wild-type βc mrna in 5c.
two other independent clones of wild-type βc, c10e and fc <dig>  showed higher mrna levels than 5c.
we next added  <dig> -dichlorobenzimidazolen1-β-d-ribofuranoside , a transcription inhibitor, to me and 5c cells and determined the steady-state amounts of wild-type βc and βc <dig> transcripts over time.
wild-type transcripts  decreased to 50% of the initial level after two hours, while βc <dig> transcripts  in me fell to half the initial level after one hour .
the reduction of βc <dig> transcripts was significantly greater than that of wild-type βc at the two and three hour time points.
given that intron v was not spliced out in ba/f <dig> transfectants, we hypothesized that the presence of  <dig> ptcs in βc <dig> mrna upstream of the pireshyg2-derived ivs resulted in nmd induction.
upregulation of βc <dig> transcripts by a translation inhibitor is greater than that of wild-type βc
it was shown that the treatment of cells with translation inhibitors result in abrogation of nmd that occurs in a translation-dependent manner  <cit> .
therefore, to examine whether nmd is implicated in the degradation of βc <dig> mrna, me cells were incubated with various concentrations of puromycin, a translation inhibitor.
the amounts of βc <dig> transcripts relative to those of gapdh increased in a dose-dependent manner, and the maximum level  was observed with  <dig> μg/ml puromycin .
emetin and cycloheximide , two other inhibitors, also induced a dose-dependent increase in βc <dig> transcripts and maximum effects similar to those induced by puromycin  .
to compare the effects of the translation inhibitor between βc <dig> and wild-type βc, me and 63m clones expressing βc <dig> and clone 5c expressing wild-type βc were incubated with  <dig> μg/ml puromycin, and βc-specific transcripts were quantified at various time points .
a time-dependent increase in βc-specific transcripts was observed in all three clones.
at  <dig>  hour time point, the increase of βc <dig> transcripts in both me  and 63m  became significantly greater than that of wild-type transcripts in 5c .
the different up-regulatory response between βc <dig> and wild-type βc was confirmed in two other clones of βc <dig> and wild-type βc .
among four βc79-expressing clones, there was no significant difference in the increase of βc <dig> transcripts induced by puromycin.
after treatment for  <dig> hours, the βc <dig> mrna levels in me and 63m further increased to  <dig>  ±  <dig> -fold and  <dig>  ±  <dig> -fold, respectively.
in contrast, there was only an  <dig>  ±  <dig> -fold increase in wild-type βc transcripts in 5c.
the resulting mrna levels of βc <dig> in me and 63m became 66% and 26% of wild-type βc, respectively .
presented as average values and s.e.m.
from three to eight independent experiments
to confirm that the effects of nmd inhibition were greater on βc <dig> than on wild-type βc, sirna targeting upf <dig>  an essential component of the nmd machinery, was used in me and 5c cells.
in both upf <dig> sirna-treated me and 5c cells, the upf <dig> rna levels became 25-30% those of the control sirna-treated cells after  <dig> hours, and 50-55% after  <dig> hours.
as shown in figure 4c, the reduction in upf <dig> for  <dig> hours resulted in a  <dig>  ±  <dig> -fold up-regulation of βc <dig> transcripts in me, which was significantly greater than that of wild-type βc in 5c .
vector sensitizes both wild-type βc and βc <dig> to nmd
although wild-type βc transcripts were less affected by nmd inhibition than βc <dig> transcripts, treatment with puromycin always resulted in a  <dig> to 12-fold increase in the amount of wild-type βc transcripts in ba/f <dig> cells.
interestingly, pireshyg <dig> provides an ivs, an ires and a hygromycin resistance gene downstream of the normal termination codon in wild-type βc.
we wondered whether the structural characteristics of pireshyg <dig> might cause nmd in wild-type βc.
to test this hypothesis, cdna of the wild-type βc was cloned into a monocistronic pmt <dig> vector that did not have them between its cloning site and poly region , and stably expressed in ba/f <dig> cells.
in contrast to what we observed when βc was cloned into pireshyg <dig> , transcripts of wild-type βc cloned into pmt <dig> showed only a slight increase  after incubation with puromycin for  <dig>  hours.
this difference was highly significant  .
the sirna-mediated upf <dig> knockdown also resulted in a  <dig>  ±  <dig> -fold change in the wild-type βc in pmt <dig>  which was significantly lower than that in pireshyg <dig>  .
when βc <dig> was cloned into pmt <dig> instead of pireshyg <dig>  the up-regulatory response of βc <dig> diminished to  <dig>  ±  <dig> -fold, which was not significantly different from that of wild-type βc cloned into pmt <dig> .
we next examined whether pireshyg2-dependent nmd affected the expression of the second cistron.
the transcripts of hygromycin resistance gene  in the cells expressing wild-type βc was increased  <dig>  ±  <dig> -fold  by puromycin treatment for  <dig>  hours.
the increase of hygr transcripts in βc <dig> clones was  <dig>  ±  <dig> -fold , which was significantly greater than that in wild-type cells .
replacement of intron v with a single nucleotide or elimination of the intron preserving a 5' single ptc fail to alter nmd effects on βc79
to examine whether the greater nmd activity on βc <dig> was attributable to intron v retained in βc <dig>  the  <dig> nucleotides of intron v were replaced by a single guanylate residue as indicated by "g" in figure 6a.
although the  <dig> nucleotides of the intronic sequence had been deleted in the resulting cdna , βc-g still retained the same  <dig> ptcs as βc <dig> .
βc-g was cloned into ireshyg <dig> and stably expressed in ba/f <dig> cells.
as shown in figure 6b, puromycin treatment for  <dig>  hours increased βc-g transcripts  <dig>  ±  <dig> -fold, which was significantly higher than the increase in 5c  but not different from that in me .
this result suggests that the presence of intron v per se is not directly involved in mediating greater nmd on βc <dig> than on wild-type βc.
we next wanted to test whether preservation of the first ptc mutation  in a variant of βc <dig> in which intron v had been completely deleted still conferred nmd to the expressed transcripts .
the resulting cdna, βc <dig>  was stably expressed using pireshyg <dig>  the up-regulatory effect of puromycin on βc <dig>  was not significantly different from that on βc <dig> .
lastly, we examined whether the pireshyg2-dependent nmd was specific to βc.
cdnas of the wild type and a truncated form  of mouse erythropoietin receptor , which are shown in figure 6c, were subcloned into pireshyg <dig> and stably expressed in ba/f <dig>  the puromycin treatment for  <dig>  hours increased wild-type epor transcripts  <dig>  ±  <dig> -fold.
in contrast, epor <dig> was increased  <dig>  ±  <dig> -fold, which was significantly greater than that of wild type .
.
discussion
this study reveals that an expression vector can destabilize the mrna of its carrying gene via nmd resulting in minimized expression levels.
when βc <dig> and wild-type βc were stably expressed using a commercial expression vector, pireshyg <dig>  the mrna levels of βc <dig> were less than one tenth those of wild-type βc.
mrna expression levels are regulated through transcriptional and posttranscriptional control mechanisms  <cit> , and mrna stability is determined by posttranscriptional regulation, which varies from one mrna species to another  <cit> .
the diversity in the transcriptional activities can be due to their integration sites  <cit> .
however, the low βc <dig> expression was uniformly seen in all clones tested, and the transcripts of βc <dig> decayed faster than those of wild-type βc.
these indicate that the posttranscriptional mechanism was involved in the low expression levels of βc <dig> mrna.
the rt-pcr indicated that 79-base intron v was retained in the βc <dig> transcripts, generating  <dig> ptcs and longer 3' utr in βc <dig> than in wild-type.
since transcripts with longer 3'utr are more sensitive to nmd in mammalian cells  <cit> , we examined whether destabilization of βc <dig> mrna via nmd caused the suppression of βc <dig> expression levels.
the faster decay of βc <dig> mrna is attributable to greater effects of nmd on βc <dig> than on wild-type βc.
since nmd is a translation-dependent process, translation inhibitors have been used to abrogate nmd, and the increase in mrna levels in the presence of a translation inhibitor is considered to reflect the effects of nmd  <cit> .
the up-regulation of βc <dig> transcripts by three different translation inhibitors indicated that βc <dig> mrna was degraded by nmd.
furthermore, the up-regulatory effects of the translation inhibition were significantly greater on βc <dig> than on wild-type βc, which diminished the difference in mrna levels between βc <dig> and wild-type βc.
the greater effects of nmd on βc <dig> were also confirmed by sirna-mediated nmd inhibition.
transcripts of βc <dig> were thus degraded more efficiently by nmd than those of wild-type βc, which resulted in lower mrna levels of βc <dig>
wild-type βc was also sensitive to nmd when expressed by pireshyg <dig>  because both translation inhibition and upf <dig> knockdown increased transcripts of wild-type βc cloned into pireshyg <dig>  these up-regulatory effects were abrogated when pmt <dig> was used instead of pireshyg <dig>  this observation indicates that the vector sensitized wild-type βc to nmd.
because βc <dig> also became insensitive to nmd in pmt <dig>  we concluded that the ires vector rendered its first cistronic genes sensitive to nmd.
there may be additional determinants for the stronger nmd to βc <dig> in the pireshyg <dig> system.
although the nucleotide in the position immediately downstream of the termination codon was shown to enhance the up-regulatory effects of translation inhibition in the order g < u, a < c  <cit> , it does not seem relevant to the difference between βc <dig> and the wild-type βc, because the nucleotide following the first ptc in βc <dig> and the one after the normal stop codon in wild-type βc are both g. the replacement of the intron v with a single nucleotide did not alter the up-regulatory effects of nmd inhibition, suggesting that the intron v was not responsible for greater nmd effects on βc <dig>  the βc <dig>  which had a single ptc at a position similar to the distal 5' ptc in βc <dig> but no frameshift, elicited a degree of nmd similar to that of βc <dig>  indicating that the 5' single ptc is sufficient to induce greater nmd effects on βc <dig>  this is comparable to recent reports in which more efficient nmd was induced when a ptc was generated closer to 5'-end resulting in longer 3'utr length  <cit> .
lastly, it is unlikely that pireshyg2-related nmd induction is specific to βc cdna.
when wild-type epor was stably expressed in the same cell line using pireshyg <dig>  upregulation of its transcripts was induced by puromycin, suggesting that wild-type epor in pireshyg <dig> vector was also a target of nmd.
the effects of puromycin were enhanced on epor <dig> that possessed longer 3'utr.
thus, the expression levels of genes encoding truncated mutants, in which ptcs generate longer 3'utrs, can be significantly reduced via vector-dependent nmd compared with wild-type genes that generally have shorter 3'utr.
CONCLUSIONS
we show here for the first time the nmd effects associated with an expression vector in a mouse hematopoietic cell line.
although the ires vector may be an interesting system for studying nmd, our study warns us that the choice of the ires vector leads to undesirable consequences especially when maximum expression of a truncated mutant with a long 3'utr is required in mammalian cells.

 expression vectors containing an internal ribosome entry site element have been widely used as bicistronic vectors that provide co-expression of two unrelated reading frames from a single transcript unit. a reading frame in a multiple cloning site downstream of a promoter is called the first cistron, and the second cistron is downstream of an ires element. pireshyg is a bicistronic expression vector that possesses an intervening sequence between the first cistron and an ires element derived from encephalomyocarditis virus, and a hygromycin resistance gene in the second cistron, which serves as a selection marker for stable transfection. it has been shown that the first cistron gene is expressed at levels comparable to those achieved in a monocistronic vector and initiation of translation is cap-dependent. however, the present study is the first to show that the use of pireshyg affects the mrna stability of their carrying genes in mammalian cells, potentially leading to their insufficient expression. nonsense-mediated mrna decay is a post-transcriptional mrna quality control system that eliminates aberrant mrnas harboring premature termination codons within protein coding regions in eukaryotes to protect the cells from accumulation of harmful or nonfunctional c-terminally truncated polypeptides. the degradation occurs in a translation-dependent manner when translation is initiated in an mrna cap-dependent manner. in mammalian cells, two determinants have been identified that distinguish "premature" termination codons from "normal" termination codons and provide a protective advantage to the normal termination codon. one is the presence of an exon-junction complex more than nucleotides downstream of a termination codon. induction of nmd requires the association between the ejc and the protein complex bound to the ribosome stalled at a ptc, which contains essential proteins to trigger nmd such as upf eukaryotic release factors, and smg. because normal termination codons generally reside either in the final exon or within nucleotides upstream of the 3'-end in the penultimate exon, the transcripts coding wild-type proteins are able to escape nmd. another determinant is the distance between the stop codon and a poly region. normal termination requires the interaction between the terminating ribosomal complex and the poly-binding proteins, which leads to faster release of a terminating ribosome from mrna. a ribosomal complex at a ptc fails to interact with pabps because of the relatively longer distance from the poly region, resulting in prolonged association with mrna, which stimulates nmd. recently, it has been reported that the length between a termination codon and poly region affects nmd efficiency, showing that longer 3'utr induces greater nmd activity. recently, we identified a novel splice variant of the granulocyte-macrophage colony-stimulating factor receptor β chain in patients with myelodysplastic syndrome, a clonal hematopoietic disorder. the splice variant retained the 79-base intron v, resulting in a frameshift that introduced eighteen ptcs downstream of the retained intron. when the cdnas encoding the βc or wild-type βc were cloned into the first cistron of pireshyg vector and stably expressed in a murine hematopoietic cell line, ba/f βc mrna levels were significantly reduced compared with those observed for wild-type βc. we show here that the pireshyg vector sensitizes the first cistron genes to nmd and, interestingly, that βc with ptcs was far more sensitive to the vector-dependent nmd resulting in minimized expression levels of the ptc-harboring gene.  intron v is not spliced out in βc clones to determine whether the intron v was spliced in the cells stably expressing βc we analyzed the presence of intron v in the integrated dna and respective rna isolated from each clone by pcr. the locations of the sequences corresponding to the primers are shown in figure and table pcr with 2s/2a primers that flank intron v distinguished βc cdna from wild-type βc cdna by the larger fragment size. in rna from all βc clones, only the larger size of pcr products was detectable. the absence of a 387-bp band suggested that intron v in βc mrna was not spliced out in ba/f transfectants resulting in the generation of ptcs. the unspliced intron v in βc mrna was also detected by pcr using a 3s primer that was complementary to the 5'-end of intron v. all rna samples without reverse transcription failed to generate any pcr products. *, the nucleotide position in the intron v-derived sequence βc mrna decays faster than wild-type βc mrna we determined the amounts of integrated dna and respective transcripts for wild-type βc or βc by real-time pcr with 1s/1a primers. compared with a clone expressing wild-type βc 5c, βc clones me and m had similar amounts of integrated βc cdna, and about half of that amount was found in clones m and 63m. however, as shown in figure 2b, in all four βc clones, rna levels for βc were less than 10% of that measured for wild-type βc mrna in 5c. two other independent clones of wild-type βc, c10e and fc showed higher mrna levels than 5c. we next added -dichlorobenzimidazolen1-β-d-ribofuranoside, a transcription inhibitor, to me and 5c cells and determined the steady-state amounts of wild-type βc and βc transcripts over time. wild-type transcripts decreased to 50% of the initial level after two hours, while βc transcripts in me fell to half the initial level after one hour. the reduction of βc transcripts was significantly greater than that of wild-type βc at the two and three hour time points. given that intron v was not spliced out in ba/f transfectants, we hypothesized that the presence of ptcs in βc mrna upstream of the pireshyg2-derived ivs resulted in nmd induction. upregulation of βc transcripts by a translation inhibitor is greater than that of wild-type βc it was shown that the treatment of cells with translation inhibitors result in abrogation of nmd that occurs in a translation-dependent manner. therefore, to examine whether nmd is implicated in the degradation of βc mrna, me cells were incubated with various concentrations of puromycin, a translation inhibitor. the amounts of βc transcripts relative to those of gapdh increased in a dose-dependent manner, and the maximum level was observed with μg/ml puromycin. emetin and cycloheximide, two other inhibitors, also induced a dose-dependent increase in βc transcripts and maximum effects similar to those induced by puromycin. to compare the effects of the translation inhibitor between βc and wild-type βc, me and 63m clones expressing βc and clone 5c expressing wild-type βc were incubated with μg/ml puromycin, and βc-specific transcripts were quantified at various time points. a time-dependent increase in βc-specific transcripts was observed in all three clones. at hour time point, the increase of βc transcripts in both me and 63m became significantly greater than that of wild-type transcripts in 5c. the different up-regulatory response between βc and wild-type βc was confirmed in two other clones of βc and wild-type βc. among four βc79-expressing clones, there was no significant difference in the increase of βc transcripts induced by puromycin. after treatment for hours, the βc mrna levels in me and 63m further increased to ± -fold and ± -fold, respectively. in contrast, there was only an ± -fold increase in wild-type βc transcripts in 5c. the resulting mrna levels of βc in me and 63m became 66% and 26% of wild-type βc, respectively. presented as average values and s.e.m. from three to eight independent experiments to confirm that the effects of nmd inhibition were greater on βc than on wild-type βc, sirna targeting upf an essential component of the nmd machinery, was used in me and 5c cells. in both upf sirna-treated me and 5c cells, the upf rna levels became 25-30% those of the control sirna-treated cells after hours, and 50-55% after hours. as shown in figure 4c, the reduction in upf for hours resulted in a ± -fold up-regulation of βc transcripts in me, which was significantly greater than that of wild-type βc in 5c. vector sensitizes both wild-type βc and βc to nmd although wild-type βc transcripts were less affected by nmd inhibition than βc transcripts, treatment with puromycin always resulted in a to 12-fold increase in the amount of wild-type βc transcripts in ba/f cells. interestingly, pireshyg provides an ivs, an ires and a hygromycin resistance gene downstream of the normal termination codon in wild-type βc. we wondered whether the structural characteristics of pireshyg might cause nmd in wild-type βc. to test this hypothesis, cdna of the wild-type βc was cloned into a monocistronic pmt vector that did not have them between its cloning site and poly region, and stably expressed in ba/f cells. in contrast to what we observed when βc was cloned into pireshyg, transcripts of wild-type βc cloned into pmt showed only a slight increase after incubation with puromycin for hours. this difference was highly significant. the sirna-mediated upf knockdown also resulted in a ± -fold change in the wild-type βc in pmt which was significantly lower than that in pireshyg. when βc was cloned into pmt instead of pireshyg the up-regulatory response of βc diminished to ± -fold, which was not significantly different from that of wild-type βc cloned into pmt. we next examined whether pireshyg2-dependent nmd affected the expression of the second cistron. the transcripts of hygromycin resistance gene in the cells expressing wild-type βc was increased ± -fold by puromycin treatment for hours. the increase of hygr transcripts in βc clones was ± -fold, which was significantly greater than that in wild-type cells. replacement of intron v with a single nucleotide or elimination of the intron preserving a 5' single ptc fail to alter nmd effects on βc79 to examine whether the greater nmd activity on βc was attributable to intron v retained in βc the nucleotides of intron v were replaced by a single guanylate residue as indicated by "g" in figure 6a. although the nucleotides of the intronic sequence had been deleted in the resulting cdna, βc-g still retained the same ptcs as βc. βc-g was cloned into ireshyg and stably expressed in ba/f cells. as shown in figure 6b, puromycin treatment for hours increased βc-g transcripts ± -fold, which was significantly higher than the increase in 5c but not different from that in me. this result suggests that the presence of intron v per se is not directly involved in mediating greater nmd on βc than on wild-type βc. we next wanted to test whether preservation of the first ptc mutation in a variant of βc in which intron v had been completely deleted still conferred nmd to the expressed transcripts. the resulting cdna, βc was stably expressed using pireshyg the up-regulatory effect of puromycin on βc was not significantly different from that on βc. lastly, we examined whether the pireshyg2-dependent nmd was specific to βc. cdnas of the wild type and a truncated form of mouse erythropoietin receptor, which are shown in figure 6c, were subcloned into pireshyg and stably expressed in ba/f the puromycin treatment for hours increased wild-type epor transcripts ± -fold. in contrast, epor was increased ± -fold, which was significantly greater than that of wild type.. discussion this study reveals that an expression vector can destabilize the mrna of its carrying gene via nmd resulting in minimized expression levels. when βc and wild-type βc were stably expressed using a commercial expression vector, pireshyg the mrna levels of βc were less than one tenth those of wild-type βc. mrna expression levels are regulated through transcriptional and posttranscriptional control mechanisms, and mrna stability is determined by posttranscriptional regulation, which varies from one mrna species to another. the diversity in the transcriptional activities can be due to their integration sites. however, the low βc expression was uniformly seen in all clones tested, and the transcripts of βc decayed faster than those of wild-type βc. these indicate that the posttranscriptional mechanism was involved in the low expression levels of βc mrna. the rt-pcr indicated that 79-base intron v was retained in the βc transcripts, generating ptcs and longer 3' utr in βc than in wild-type. since transcripts with longer 3'utr are more sensitive to nmd in mammalian cells, we examined whether destabilization of βc mrna via nmd caused the suppression of βc expression levels. the faster decay of βc mrna is attributable to greater effects of nmd on βc than on wild-type βc. since nmd is a translation-dependent process, translation inhibitors have been used to abrogate nmd, and the increase in mrna levels in the presence of a translation inhibitor is considered to reflect the effects of nmd. the up-regulation of βc transcripts by three different translation inhibitors indicated that βc mrna was degraded by nmd. furthermore, the up-regulatory effects of the translation inhibition were significantly greater on βc than on wild-type βc, which diminished the difference in mrna levels between βc and wild-type βc. the greater effects of nmd on βc were also confirmed by sirna-mediated nmd inhibition. transcripts of βc were thus degraded more efficiently by nmd than those of wild-type βc, which resulted in lower mrna levels of βc wild-type βc was also sensitive to nmd when expressed by pireshyg because both translation inhibition and upf knockdown increased transcripts of wild-type βc cloned into pireshyg these up-regulatory effects were abrogated when pmt was used instead of pireshyg this observation indicates that the vector sensitized wild-type βc to nmd. because βc also became insensitive to nmd in pmt we concluded that the ires vector rendered its first cistronic genes sensitive to nmd. there may be additional determinants for the stronger nmd to βc in the pireshyg system. although the nucleotide in the position immediately downstream of the termination codon was shown to enhance the up-regulatory effects of translation inhibition in the order g < u, a < c, it does not seem relevant to the difference between βc and the wild-type βc, because the nucleotide following the first ptc in βc and the one after the normal stop codon in wild-type βc are both g. the replacement of the intron v with a single nucleotide did not alter the up-regulatory effects of nmd inhibition, suggesting that the intron v was not responsible for greater nmd effects on βc the βc which had a single ptc at a position similar to the distal 5' ptc in βc but no frameshift, elicited a degree of nmd similar to that of βc indicating that the 5' single ptc is sufficient to induce greater nmd effects on βc this is comparable to recent reports in which more efficient nmd was induced when a ptc was generated closer to 5'-end resulting in longer 3'utr length. lastly, it is unlikely that pireshyg2-related nmd induction is specific to βc cdna. when wild-type epor was stably expressed in the same cell line using pireshyg upregulation of its transcripts was induced by puromycin, suggesting that wild-type epor in pireshyg vector was also a target of nmd. the effects of puromycin were enhanced on epor that possessed longer 3'utr. thus, the expression levels of genes encoding truncated mutants, in which ptcs generate longer 3'utrs, can be significantly reduced via vector-dependent nmd compared with wild-type genes that generally have shorter 3'utr.  we show here for the first time the nmd effects associated with an expression vector in a mouse hematopoietic cell line. although the ires vector may be an interesting system for studying nmd, our study warns us that the choice of the ires vector leads to undesirable consequences especially when maximum expression of a truncated mutant with a long 3'utr is required in mammalian cells. 
compared with wild-type βc, the mrna levels of βc were less than one tenth and decayed faster. both translation inhibition and upf knockdown led to significantly greater up-regulation of βc than wild-type βc. however, the use of a monocistronic pmt vector abolished the up-regulatory effects of translation inhibition and upf knockdown on both wild-type βc and βc suggesting that the nmd is attributable to a structural determinant in pireshyg the elimination of the intron and the proximal 3' ptcs did not alter the greater effects of translation inhibition on βc suggesting that the first ptc, which determines 3'utr length, was sufficient to enhance nmd efficiency. thus, transcripts of ptc-harboring genes with longer 3'utr are more efficiently degraded by the vector-dependent nmd than those of wild-type genes with relatively shorter 3'utr, resulting in minimized expression of truncated mutants. however, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mrna decay, an mrna surveillance system that degrades mrna with a premature termination codon. pireshyg provides the first cistron gene with a long 3'utr consisting of a downstream intervening sequence, an internal ribosomal entry site and the second cistron. therefore, we hypothesized that the first cistron genes in pireshyg are sensitized to nmd, which affects their expression levels. in mammalian cells, the induction of nmd requires either a long 3'utr or the presence of an exon-junction complex downstream of a ptc. to examine this hypothesis, cdnas encoding human granulocyte-macrophage colony-stimulating factor receptor β chain and its splice variant, in which the retention of a 79-base intron caused a frameshift generating ptcs, were cloned into pireshyg and stably expressed in a murine cell line, ba/f pireshyg has been widely used as a bicistronic expression vector.
 pireshyg has been widely used as a bicistronic expression vector. however, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mrna decay, an mrna surveillance system that degrades mrna with a premature termination codon. in mammalian cells, the induction of nmd requires either a long 3'utr or the presence of an exon-junction complex downstream of a ptc. the efficiency of nmd is greater when a ptc generates longer 3'utr. pireshyg provides the first cistron gene with a long 3'utr consisting of a downstream intervening sequence, an internal ribosomal entry site and the second cistron. therefore, we hypothesized that the first cistron genes in pireshyg are sensitized to nmd, which affects their expression levels. to examine this hypothesis, cdnas encoding human granulocyte-macrophage colony-stimulating factor receptor β chain and its splice variant, in which the retention of a 79-base intron caused a frameshift generating ptcs, were cloned into pireshyg and stably expressed in a murine cell line, ba/f  compared with wild-type βc, the mrna levels of βc were less than one tenth and decayed faster. both translation inhibition and upf knockdown led to significantly greater up-regulation of βc than wild-type βc. however, the use of a monocistronic pmt vector abolished the up-regulatory effects of translation inhibition and upf knockdown on both wild-type βc and βc suggesting that the nmd is attributable to a structural determinant in pireshyg the elimination of the intron and the proximal 3' ptcs did not alter the greater effects of translation inhibition on βc suggesting that the first ptc, which determines 3'utr length, was sufficient to enhance nmd efficiency. thus, transcripts of ptc-harboring genes with longer 3'utr are more efficiently degraded by the vector-dependent nmd than those of wild-type genes with relatively shorter 3'utr, resulting in minimized expression of truncated mutants.  we conclude that pireshyg which sensitizes its bicistronic transcripts to nmd, may be useful for studying nmd but should be avoided when maximum expressions of ptc-harboring genes are required. 