it is usually controlled by cis - acting factors , such as au - rich elements ( ares ) in the 3utr , and transacting factors , such as rna - binding proteins , mirna , and ribonucleases ( reviewed in refs .
traditionally , the 3utr has been viewed as the region most recognized by rna - binding proteins such as tristetraprolin ( ttp / zfp36 ) and hur , and that exoribonucleases largely mediate mrna decay .
conversely , very few mammalian endoribonucleases are known and they were shown to cleave at ua di - ribonucleotides .
rnase l  has a substrate cleavage preference at uu and ua in single - stranded viral mrnas during acute viral infections and possibly in cellular mrnas under certain conditions .
another endoribonuclease has been described that preferentially cleaves single - stranded mrna - coding regions inside ua and ug in several mrnas , including c - myc , mdr1 , and -globin .
since ua and uu dinucleotides are common targets for these known endoribonucleases , we sought to eliminate , or at least reduce , uw ( w = a or u ) dinucleotides in the coding region of several genes , without affecting the protein code .
the ua dinucleotides are generally under - represented in human and other vertebrate genomes and are further deficient in the mrna when compared to nontranscribed dna .
deficiency of uw frequency in coding regions may impose a selection against endoribonuclease - mediated mrna decay inside ua dinucleotides . in this report
, we showed that mrna stability increased as a result of reduction in uw frequency , leading to increased protein production .
this observation suggests a general approach for rational design of synthetic genes to yield robust protein expression .
the uw frequency was reduced in synthetic genes by choosing alternative codons that do not contain uw , as well as by replacing uw in the dicodon boundary by changing the first codon ( figure 1a ) .
several coding regions were utilized to assess the approach , namely the reporter genes enhanced green fluorescent protein ( egfp ) and luciferase and the biotechnology related genes interferon- and hepatitis b surface antigen ( hbsag ; figure 1b ) .
the modification resulted in a significant reduction of uu dinucleotides ( > 68% , p < 0.001 , -test ) .
the ua and , to a lesser extent , au dinucleotides were reduced as a result of the sequence modification , whereas ga dinucleotide frequency ( used as a control ) did not decrease ( figure 1b ) .
the mammalian expression vectors harboring these sequences were cotransfected with red fluorescent protein plasmid into the hek293 cell line , and then the protein expression levels were assessed in comparison to unmodified sequences ( figure 1c ) .
there was increased protein expression ( five- to eight - fold ) from expression constructs with uw - reduced coding regions . when inserted , with red fluorescent protein plasmid also , into the cho1 cell line , which is a biotechnology standard cell line , the modified expression cassettes also gave higher protein expression ( figure 1d ) .
thus , the data demonstrate that uw reduction in coding regions leads to higher protein expression . in order to evaluate the effect of sequence modification on dna uptake as a factor in transfection efficiency , we cotransfect the different expression vectors with a rhodamine - conjugated linear polyethylenimine derivative .
we found no statistically significant difference in dna uptake between the wild type and the sequence modified uw - reduced constructs ( supplementary figure s2 ) .
a marginal significance ( p = 0.06 ) was observed for the uw - reduced hbsag construct , but this small increase ( 19% increase ) played only a little role in the substantial increase in protein expression ( 400% , figure 1c ) .
the polyethylenimine - mediated transfection of wt and uw - reduced egfp gave comparable results to liposomal - mediated transfection in that uw - reduced egfp showed increased expression ( supplementary figure s1 ) .
next , we focused on egfp so that we could study both mrna and protein expression ( by fluorescence ) . a representative image of wt egfp and uw - reduced egfp shows a dramatically enhanced fluorescence , whereas no increase is shown for the cotransfected red fluorescent protein , plasmid ( figure 2a and b ) .
expression of the uw - reduced luciferase - coding region under conditions of low amounts of transfected dna ( 10 ng / well ) was higher when compared to 75 ng / well ( figure 2c ) .
the data in figure 2 and supplementary figure s2 , taken together , indicate that the enhancing effects of uw reduction on protein expression are not related to preferential dna uptake or to variations in transfection efficiency .
furthermore , we have examined the mrna half - life changes due to the uw sequence reduction .
we employed the one - phase exponential decay model which describes kinetics in which the levels of a substance never decay to zero but reach a plateau .
this is the case of our mrna decay curves here , which fit the model with superior goodness of fit ( r  0.95 ) .
also , others ' work applied the one - phase exponential decay model on mrna kinetics that behaves in a manner where decaying mrna levels do not reach zero but a plateau .
we found that the mrna for the uw - reduced egfp had a longer half - life ( 2.53-fold increase , p = 0.01 , f - test ) compared to the wt mrna , indicating that uw frequency reduction led to mrna stabilization ( figure 3a ) .
it is generally known that such a modest degree of mrna half - life increase can lead to a substantial enhancement of protein expression .
steady - state mrna levels were also higher ( 2.75-fold , p < 0.05 ) when expressed from the uwr - egfp construct compared to the wild - type unmodified egfp dna ( figure 3b ) . when egfp and uwr - egfp - coding regions were fused with 3utr that contained
are from the unstable tumor necrosis factor - mrna , higher protein expression was observed from the uwr - egfp construct compared to the wt egfp ( 32-fold versus 8-fold , p < 0.001 ; figure 3c ) . in general , under conditions of low mrna expression , transgene expression usually correlated positively with protein expression . in order to understand the mechanism of increased mrna stability due to uw reduction in coding regions , we first investigated the involvement of rnase l by overexpression and small interfering rna experiments .
we found that overexpression of ha - tagged rnase l did not reduce mrna stability of either egfp or uwr - egfp ( figure 4a , b ) .
western blotting demonstrate expression of transfected rnase l by probing with antibody to the ha tag ( figure 4a , b ; insets ) . at the protein level
, we also noted that rnase l did not reduce the levels of wt or uwr egfp , although there was a slight increase in wt egfp expression ( figure 4c ) .
hence , in general , we found either no effect or an inconsistent effect of rnase l overexpression or of treatment with a small interfering rna to rnase l ( data not shown ) .
we therefore resorted to another strategy that utilized the c124r zinc finger mutant form of ttp ( ttp / zfp36 ) .
the ttp binds to ares as small as uauuuau and the c124r mutant fails to bind to ares .
however , c124r can still interact with a number of general mrna decay enzymes such as those involved in deadenylation ( e.g. , caf1 ) , decapping , and exonuclease activity .
this dominant negative activity appears to be specific to the 3utr - are at lower concentrations , but we hypothesized that it may have broader effects at higher concentrations in a manner independent of the 3utr .
thus , we overexpressed c124r ttp in hela tet - off cell line and studied the effects on uw - reduced egfp mrna and wild - type mrna .
western blotting confirmed expression of the transfected c124r ttp ( figure 4d , e insets ) .
the mrna half - life was assessed for both wt egfp and uwr egfp transcripts released from constructs under regulation of tet - off cytomegalovirus promoter
. the dominant negative effect of c124r ttp , as demonstrated by mrna stabilization , was clearly observed in the case of the response of wt egfp mrna turnover ( figure 4d ) .
further increases in uwr egfp mrna stabilization were not observed as a consequence of the dominant negative effect of c124r ttp ( figure 4e ) .
subsequently , no increase in protein production ( fluorescence ) from uwr egfp mrna was observed in response to the dominant negative effect of c124r ( figure 4f , middle columns ) .
in contrast , wt egfp protein levels were affected by the stabilizing effect of c124r on wt egfp mrna ( figure 4f , left columns ) .
in addition to tumor necrosis factor- mrna , c124r ttp ( but not wild - type ttp ) has been shown recently to increase an array of au - rich mrnas such as upa , upa receptor , nf - kappa b inhibitor , matrix metalloproteinase-1 , and interleukin-10 .
the c124r has the ability to increase protein expression from uwr egfp transcripts only when the uwr egfp - coding region is fused with are-3utrs , such as those of tumor necrosis factor- ( figure 4f , right columns ) .
this aspect may lead to an improved use of uw - reduced reporter genes such as egfp and luciferase , which is post - transcriptional effect of gene expression specific to the 3utr .
the present approach differs from the classical codon optimization that depends on species - specific bias of codon usage , and it is thought to be a natural selection for translational optimization .
this served as the basis for improving overexpression of proteins in host cells of a different species .
our approach is different : first , unlike classical codon bias optimization , both codon and dicodon boundaries are considered here .
second , this rationalized approach is based on reduction of uw dinucleotides ; these dinucleotides are rare in mammalian coding regions and have been shown to correlate with accelerated mrna decay .
our approach has led to increased mrna stabilization and , subsequently , increased protein expression levels .
however , other possible post - transcriptional effects , such as translation , are likely to take part as well .
validation of this role of modification on translation efficiency would require a different set of experiments .
the very modest increase ( for example , a 14% increase ) in mrna stability observed with classical codon optimization may possibly be due to an inherited partial uw reduction .
for example , the preferred human codon for isoleucine is auc rather than auu or aua .
overall , by resisting mrna decay using reduced uw frequency and elimination of uw in dicodon boundaries , this rationalized approach may find utility as a general method to boost recombinant protein production in mammalian cells .
the uw reduction approach may find broader applications if confirmed in other expression systems , such as those based on bacterial and insect cells .
also , the approach remains to be tested with stable cell lines expressing the uw - reduced coding regions , as unlike transient transfection , potential regulatory mechanisms such as dna methylation may adversely affect stable transgene expression . the approach can enhance reporter genes for difficult to transfect cells or can be used to study 3 utr - mediated post - transcriptional effects due to instability sequences , such as ares , or mirna action .
synthetic biology . coding regions of egfp , luciferase , interferon 2a , and hbsag were modified according the following algorithm : ( i ) by exchanging uw - containing codons with synonymous codons that lack or reduce uw ( supplementary table s1 ) and ( ii ) by destroying the uw dicodon boundary by exchanging the first codon with synonymous codons ( supplementary table s2 ) . the modified coding region sequences ( supplementary table s3 )
were synthesized by geneart ( regensburg , germany ) and all constructs , including wild - type coding regions , weresubcloned using sali and bamh1 sites in the same vector ( gwiz ; genlantis , san diego , ca ) .
the vector is expressed under control of the cytomegalovirus / intron a promoter and with the stable bovine growth hormone 3utr .
cloning of the are derived from the tumor necrosis factor- mrna 3utr was previously described .
dinucleotide frequency analysis was executed by the compseq program , an emboss algorithm that calculates nucleotide composition in a given sequence ( http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::compseq ) .
the formula of observed ( 0 ) over expected ( e ) frequency ratio is as follows : ( xy o / e = dxy / nxny , where nx denotes the frequency of nucleotide x , and dxy is the frequency of the dinucleotide ( xy ) .
cell lines and transfection . the hek293 and cho1-k cell lines were obtained from american type culture collection ( rockville , md ) and cultured in dmem medium ( invitrogen , carlsbad , ca ) or dmem / f12 ( in case of cho1-k ) supplemented with 10% fbs and antibiotics .
the tet - off hela cell line , obtained from clontech ( mountain view , ca ) , was maintained in dmem with selection drug g418 ( sigma , st louis , mo ) .
cells were transfected with vectors using lipofectamine 2000 reagent ( invitrogen ) according to the manufacturer 's instructions .
all transfections were performed in several replications , and our intrawell transfection efficiency was < 6% variation . the red fluorescent protein expression vector ( evrogen , san diego , ca )
was used at  ratio of the test plasmid concentration for the purpose of normalization . for rna experiments , egfp ( 50 ng per 1  10 cells ) was used for monitoring transfection efficiency .
luciferase expression was assessed using the luciferase assay system from promega ( madison , wi ) and a luminometer .
interferon- and hbsag was quantified by enzyme - linked immunosorbent assay ( elisa ) obtained from pbl ( piscataway , nj ) and abzyme ( piscataway , nj ) .
for transfected cells with c124r ttp expression vector ( a gift from dr perry j. blackshear , nih , us ) and ha - tagged rnase l expression vector ( synthesized by geneart ) , western blottings were performed to confirm the protein expression from the vectors .
sds gels ( protogel ; national diagnostics , atlanta , ga ) followed by transfer to nitrocellulose membranes ( hybond ecl ; amersham biosciences , piscataway , nj ) .
membranes were hybridized with primary antibody to goat anti - ttp ( 1/500 ) ( santa cruz biotech , santa cruz , ca ) , rat anti - ha a ( 1/50 ) ( roche , basel , switzerland ) , or mouse anti - gapdh ( 1/500 ; abcam , cambridge , uk ) followed by the appropriate secondary horseradish peroxidase -conjugated antibody .
signal detection was performed with ecl western blotting detection reagents ( amersham biosciences , amersham , uk ) .
the fluorescence intensity was assessed using high - resolution images obtained from an automated laser - focus high - throughput bd pathway 435 imager ( bd biosciences , san jose , ca ) . the variance in gfp fluorescence among replicate
the experiments were normalized with red fluorescent protein plasmid at  ratio of the test plasmid concentration .
image processing , segmentation , and fluorescence quantification were facilitated by the proxcell program previously described .
fluorescence of egfp ( excitation , 488 nm ; emission , 507 nm ) and red fluorescent protein ( excitation , 553 nm ; 574 nm ) was processed with appropriate filters of the bd imager .
data are presented as mean  sem of total fluorescence intensity in each well , with replicate readings ranging from three to four , as indicated in the text .
the student t - test was used when comparing two data groups , while analysis of variance was performed for each data set having three or more data groups .
dna uptake . the rhodamine - conjugated linear polyethylenimine derivative ( excitation at 550 nm ; emission at 575 nm ) was used to assess the uptake of dna by the wild type and modified plasmids .
cells were transfected with a complex of 0.75  l of jetpei - fluor ( polyplus , paris , france ) and 200 ng of the plasmid dna .
after 18-hour incubation , the cells were visualized by automated fluorescence microscopy and the fluorescence quantified as described above .
this protocol was also repeated in the presence of the lipofectamine 2000 to assess its compatibility during liposomal transfection and was found compatible .
reverse transcription was performed using 200 ng total rna , 500 ng oligo dt(1823 ) , 500 mmol / l dntp mixture , 20 u rnasin ( pharmacia , milton keyries , uk ) , and 200 u of superscript ii ( invitrogen ) . real - time pcr was performed using custom - made primer sets ( applied biosystems , foster city , ca ) and taqman master mix according to the manufacturer 's instructions .
the 6-carboxyfluorescein - labeled taqman hybridization probes were designed to target the junction of exons 1 and 2 of egfp or uwr egfp to minimize dna contamination .
the primer set for egfp : forward ctccatcttcgcggtagct , reverse ttcttctcctttgctagccatttct , probe : ccgccgttcagtcg ccgt .
the primer set for uwr egfp : forward ctccatcttcgcgg tagct , reverse ccatggtggcaagcttgtc , probe : ccgccgtt cagtcgccgt . as internal control , we used a vic - labeled gapdh mrna probe ( applied biosystems ) .
the primer sets failed to amplify vector dna ( negative control ) and were specific for the complementary dna .
pcr protocol was as follows : initiation steps : 2 minutes at 50 c followed by 10 minutes at 95 c , denaturing step : 15 seconds at 95 c , and annealing / extension 1 minute at 60 c .
the amounts of molecules of complementary dna were calculated using chroma 4 dna engine cycler ( bio rad , hercules , ca ) software and an eight - point standard curve using serial dilutions of purified and quantified amplicon dna .
the use of standard curve method ensures that all amplifications proceed with a comparable and high efficiency .
the lowest internal control measurement was set as 1 fold and the copy number of the wt egfp and uwr egfp were normalized .
hela tet - off cells were cotransfected with teto - regulated wt or uwr egfp constructs , along with vector , c124r ttp , or rnase l expression vector overnight , and then treated with doxycycline ( 1  g / ml ) to shutoff transcription .
the rna was extracted by the rnazol method at multiple points and then subjected to reverse transcription - qpcr as described above . to ensure equal proportions of transfected cells for each time point ,
cotransfections were performed in 10-cm culture plates using 4  g of the vector dna , and 24 hours later the cells were split into 6-well plates .
the mrna half - life was determined using a one - phase exponential decay model .
the equation y = span  exp(k  x ) + plateau describes the kinetics of mrna decay .
y starts out equal to span + plateau and decreases to plateau with a rate constant k. the half - life of the decay is 0.6932/k .
expression of wt and uwr egfp after pei - mediated transfection . click here for additional data file .
 uu and ua dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease - mediated mrna decay . 
 this study hypothesized that reducing uu and ua ( uw ) dinucleotides in the mrna - coding sequence , including the codons and the dicodon boundaries , may promote resistance to mrna decay , thereby increasing protein production . 
 indeed , protein expression from uw - reduced coding regions of enhanced green fluorescent protein ( egfp ) , luciferase , interferon- , and hepatitis b surface antigen ( hbsag ) was higher when compared to the wild - type protein expression . 
 the steady - state level of uw - reduced egfp mrna was higher and the mrna half - life was also longer . 
 ectopic expression of the endoribonuclease , rnase l , did not reduce the wild type or uw - reduced mrna . a mutant form of the mrna decay - promoting protein , tristetraprolin ( ttp / zfp36 ) , which has a point mutation in the zinc - finger domain ( c124r ) , was used . 
 the wild - type egfp mrna but not the uw - reduced mrna responded to the dominant negative action of the c124r zfp36/ttp mutant . 
 the results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells . 
Introduction
Results
Discussion
Materials and Methods
Supplementary Material