BACKGROUND
genomic data from many different organisms indicate that synonymous codon usage is typically biased.
this bias is an intriguing property that reflects a composite of different evolutionary forces such as mutational bias, genetic drift, and natural selection  <cit> .
in many species, including drosophila, one of the dominant forces affecting the bias is thought to be natural selection for translational efficiency or accuracy, or both .
the most abundant codons are preferred because of the abundance of the cognate trnas  <cit> , and thus the use of the codons maximizes the translation speed and minimizes the amino acid misincorporation rate  <cit> .
there is limited experimental evidence that major codons are translationally superior  <cit> , but empirical data can be explained well in this framework.
the selective force for translational efficiency is weak enough to allow confounding neutral processes to dominate in some cases  <cit> .
for example, the effect of genetic drift  is inferred from the comparison between d. melanogaster and d. simulans  <cit> .
other types of selective forces may also counteract translational selection at synonymous sites.
splicing regulation is one of them.
specifically, there are binding sites within exonic regions for serine-arginine-rich  proteins, which enable correct splicing.
these binding sites, known as exonic splicing enhancers , are usually located within the vicinity of potential intron-exon junctions  <cit> , and many of them are ~6– <dig> bp in length  <cit> .
these splice signals within the coding regions can potentially affect synonymous codon usage.
recent genomic scale analyses in d. melanogaster revealed that translationally optimum codons are not preferred near the intron-exon boundary, and thus are not splice optima  <cit> .
moreover, warnecke and hurst  <cit>  showed that codons putatively involved in eses are almost never translationally optimal, and therefore, a conflict exists between translational advantage and splice efficiency in codon usage.
they also showed that this conflict is larger in highly expressed genes, although the effect of expression level was not very strong.
these conclusions obtained from a large genomic scale dataset stimulated me to find a well-controlled gene system in which splicing regulation can be effectively analyzed.
alternatively spliced exons  of down syndrome cell adhesion molecule  appeared to be an excellent model system to study such regulation, because this molecule contains the largest number of ases known to date.
there are four exons that have multiple alternatively spliced forms , which produces  <dig>  distinct potential isoforms  <cit> .
the structure of the gene is shown in figure  <dig>  these ases can generate unique cell identity by expressing distinct sets of isoforms in the nervous systems where the molecule plays a crucial role in neuronal wiring .
also, the large spectrum of distinct isoforms is required in hemocytes where the molecule is necessary for effective immune response  <cit> .
thus, this molecule's unique gene structure has probably arisen from the necessity to generate a diverse repertoire of alternatively spliced variants.
in addition to the fact that dscam contains the largest number of ases known to date, these ases provide an ideal system for analyzing the effect of splicing regulation for the following reasons.
first of all, these ases are spliced out from a single pre-mrna, and therefore, splicing frequency can be separated from transcription frequency.
this kind of within-transcript comparison is the only way to analyze the effect of splicing frequency.
these ases are also under the control of the same splicing machinery within the same cell environment in which they are expressed.
second, since these ases are in the same genomic region, they are controlled for many possible confounding factors affecting synonymous codon usage such as local gc content  <cit>  and recombination rate  <cit> .
finally, these ases are similar in length and amino acid composition  <cit> , which are also factors known to influence the codon usage  <cit> .
it should be noted that most of these paralogous ases diverged prior to the d. melanogaster – d. virilis species split  <cit> , which indicates that the synonymous sites of these ases have diverged beyond saturation  <cit> .
therefore, it is reasonable to assume that their codon usage is effectively independent from each other.
additionally, all the  <dig> species of drosophila whose genomic sequences are available have diverged after the d. melanogaster – d. virilis species split  <cit> , and thus, orthologous pairs of ases can be identified in many cases.
in this study, i analyzed these ases to delineate the effect of splicing regulation on synonymous codon usage by comparing the levels of codon bias in the central and intron-exon boundary regions of these exons.
i studied the effect of two splice-related factors that affect the regional differences in cbi among these ases; the splicing frequency  and the distance from adjacent constitutive exons.
in addition, i also conducted a comparison between paralogous exonic regions of retrotransposed processed genes and their parental genes, in order to investigate the effect of splicing regulation on synonymous codon usage in different gene systems.
RESULTS
codon usage bias towards translationally superior codons is reduced near the 3' intron-exon boundaries in most of the dscam exons
genome-wide analysis in drosophila has indicated reduced codon usage bias toward translationally optimal codons within  <dig> nucleotides of an intron-exon boundary, where the vast majority of functional eses are assumed to exist  <cit> .
in order to investigate whether this trend exists in the ases of dscam exons  <dig>   <dig>  and  <dig>  i calculated codon bias index  in the center and the boundary regions and compared the differences.
following warnecke and hurst  <cit> , the boundary region was defined as the exonic region within  <dig> full codons of the boundary, excluding the partial codons at the junction, and the center region was defined as the regions remaining after subtracting the flanks .
comparisons of cbi values between the center and the boundary regions of these exon clusters are shown in figure  <dig>  cbi values of the 3' boundary region are lower than the central exonic regions in the ases of exons  <dig> and  <dig> .
among the exon  <dig> ases , there are significant differences between the center and 3' boundary  and 5' boundary and 3' boundary , but not between center and 5' boundary .
among the exon  <dig> ases , the difference between center and 3' boundary is significant , but the differences between center and 5' boundary , and 5' boundary and 3' boundary  are not significant.
other internal exons which are longer than  <dig> bp  also show significant differences between the center and 3' boundary , the center and 5' boundary , and 5' boundary and 3' boundary .
these comparisons indicate that in most of the exons in this gene, codons in the 3' boundary regions are less adapted for translation efficiency compared to codons in the central exonic regions.
since the regions analyzed were short, the reduction of cbi in the 3' boundary region could be due to bias in amino acid composition.
i compared the codon usage pattern of each amino acid in the 3' boundary region and the remaining 5' region in a pooled sample of exon  <dig> and  <dig> ases.
among  <dig> amino acids with degenerate codons,  <dig> showed higher frequencies of translationally preferred codons in the remaining region  compared to those in the 3' boundary region .
among them,  <dig> amino acids  showed statistical significance at the level of p <  <dig>  by fisher's exact test.
these results clearly indicate that the bias in amino acid composition is not the reason for the reduced cbi in the 3' boundary regions.
since warnecke and hurst  <cit>  indicated that translationally optimal codons and splice optimal codons  in d. melanogaster are mostly mutually exclusive, my data suggest stronger restriction on codon usage due to splicing regulation in the 3' boundary regions than in the 5' boundary regions throughout the gene, except in the ases of the exon  <dig> cluster.
despite the genome-wide tendency of reduced cbi in the intron-exon boundary regions  <cit> , various patterns among different genes and exons can arise at least partly by different machineries used for splicing regulation .
for example, olson et al.
<cit>  have shown that the heterogeneous nuclear ribosomal protein hrp <dig> plays a key role as a splicing repressor in mutually exclusive splicing of the dscam exon  <dig> ases, but depletion of this protein had no effect on the splicing of ases in other exon clusters.
a possible reason for the distinct codon usage pattern in the 3' boundary regions could be that some of the splicing machineries used in this gene utilize signals in these regions to a larger extent than those in other exonic regions.
specifically, the reduced cbi3' boundary regionin the ases of the exon  <dig> cluster, where the difference in cbi between the center and the 3' boundary regions is most apparent , may be explained by the unique model for the mechanism of mutually exclusive alternative splicing proposed by graveley  <cit> .
he discovered two classes of conserved elements in the non-coding regions of the dscam exon  <dig> cluster; the docking site, located in the intron downstream of constitutive exon  <dig>  and the selector sequences, which are located upstream of each exon  <dig> ase.
each selector sequence is complementary to a portion of the docking site.
the model suggests that the formation of the rna structure by docking site:selector sequence interaction is a central component of the mechanism guaranteeing that only one exon  <dig> ase is included in each dscam mrna.
this docking site:selector site interaction brings together the 3' end of exon  <dig> and the 5' end of one of the exon  <dig> ases.
if this is the case, there must be another factor that brings together the 3' end of the chosen ase and the 5' end of exon  <dig>  the upstream splice reaction aided by docking site:selector site interaction and the downstream reaction may occur simultaneously, but the latter may be where eses are mainly involved.
if so, then this could be the reason for observing stronger restriction on the codon usage in the 3' boundary regions of these exon  <dig> ases.
although obtaining a detailed picture of the splicing machineries requires further experiments, the reduction of cbi in the 3' intron-exon boundary regions in this gene suggests that splice-related selection is present.
i further examined this possibility by comparing the level of cbi reduction and experimentally obtained splicing frequency data.
conflict between translational selection and selection for splice efficiency increases with the splicing frequency
iida and akashi  <cit>  have shown that within alternatively spliced genes, gc-ending codons are more abundant in constitutive than in alternatively spliced exons in drosophila and humans.
this is consistent with the prediction that translational selection should act more strongly to bias codon usage in constitutive than in alternative exon codons because the former are translated more often than the latter.
the selection coefficient for translational selection is expected to become larger in the more frequently translated codons.
this is also the pattern that was observed in dscam when whole exonic regions of constitutive and alternative exons were compared; average cbi of  <dig> internal constitutive exons without utrs was  <dig>  ±  <dig>  , whereas that of  <dig> ases was  <dig>  ±  <dig>  .
as suggested from the comparison of cbi between constitutive and alternative exonic regions, if majority of the dscam exonic region is under translational selection codon usage in the 3' boundary regions and the remaining exonic regions should show different trends in relation to the expression level, because of the additional constraint due to splicing regulation in the former.
if the latter is relatively free from the constraint due to splicing regulation, its cbi should show a positive correlation with the expression level due to translational selection.
this can be the background level of translational selection.
whereas, if synonymous sites in the 3' boundary region are under selection for splicing efficiency, it is expected that cbi in this region should show relatively constant values or a negative correlation with the splicing frequency .
the level of conflict, which can be evaluated by the difference between the 3' boundary region and the rest of the region  should then increase with the splicing frequency.
the expression levels of multiple dscam splice variants have been studied extensively by neves et al.
<cit>  using their custom microarray.
taking advantage of the availability of their data, i analyzed the 3' boundary region of the ases in exons  <dig> and  <dig>  where cbi was significantly reduced in comparison to the remaining 5' region .
i used the relative order of the expression level of each exon  <dig> and  <dig> ase in hemocyte-derived s <dig> cell lines as the estimated order of splicing frequency .
in the case of exon  <dig>  most ases are expressed at some or all stages of development, and their expression levels showed only moderate differences among different tissues and among different developmental stages  <cit> .
thus, i used all the  <dig> ases with the expression level data  for the following analyses.
as expected, a positive correlation was detected between the expression level and Δ-cbirest – 3' boundary = cbi5' boundary + central – cbi3' boundary , and also between the expression level and the cbi of the remaining 5' region .
the correlation between the cbi3' boundary and the splicing frequency was not significant , suggesting a relatively constant level of cbi values in this region with a weak trend of negative correlation with the splicing frequency .
the contrasting expression-level dependency of codon usage between the 3' boundary and the remaining exonic regions indicates regional differences in the type of selection affecting the synonymous codon usage within exons of these exon  <dig> ases.
in particular, the positive correlation between Δ-cbirest – 3' boundary and the expression level indicates that the intensity of splice-related selection counteracting the translational selection increases with the splicing frequency.
therefore, the conflict between these two evolutionary forces in the 3' boundary region becomes more pronounced in the highly expressed  ases of the exon  <dig> cluster.
in the case of exon  <dig> ases,  <dig> or  <dig> of the  <dig> ases are spliced more frequently than others in a number of tissues  <cit> .
about half of the ases are represented at less than the overrepresentation level of  <dig>   in these tissues  <cit> .
since the expression level order of these less abundant ases is likely to be unreliable, i used only the last half of the ases ordered from the lowest to the highest expression level, where the actual trend in transcript abundance is visible from the heatmap .
using these  <dig> ases of exon  <dig>  a positive correlation between the expression level and Δ-cbirest – 3' boundary  was detected.
there was no significant correlation between the expression level and cbi of the 3' boundary region  or between the expression level and cbi of the remaining 5' region .
these results indicate that even with a smaller number of ases analyzed compared to the exon  <dig> cluster, the intensity of conflict in the exon  <dig> ases between selection for translational efficiency and for splicing efficiency also correlates with the expression level.
the distance to the adjacent constitutive exon affects codon usage in the intron-exon boundary region
among the mutually exclusive ases of this gene, the nucleotide distance to the adjacent constitutive exons is a factor, which is likely to affect the occurrence of splicing.
presumably, closer splice sites are more likely to interact.
this distance effect in ases is analogous to the effect of intron length in the constitutive exons in humans and mouse, in which exons flanked by longer introns contained a significantly higher abundance of putative eses than those flanked by shorter introns  <cit> .
this is consistent with the "exon definition" model of splicing that invokes ese oriented initial splice-site recognition  <cit> .
this model applies better to genes with small exons and large introns, which fits the case of exon  <dig> and  <dig> ases.
the exon  <dig> ases spread over a ~ <dig> kb region and the distance between splice sites varies between ~ <dig> bp to ~ <dig> kb.
despite this wide distance range, there was no apparent relationship between the position of ases relative to adjacent constitutive exons and the expression level , which suggest that the distance effect is controlled in some way, possibly by exonic signals.
it can be inferred from the docking site:selector site model that the downstream splice reaction could be the major function of the eses in the exon  <dig> ases.
if so, the intensity of these splice signals should increase in the ases closer to the upstream adjacent exon  <dig> to complement the distance effect.
inconsistent with this prediction, there was no significant positive correlation between cbi3' boundary and the sequence distance from exon  <dig> .
however, since the intensity of splicing regulation on synonymous codon usage is likely to vary with the position of amino acid residues, specific codons were examined to see if there are variations in codon usage that may contribute to complementing the distance effect.
four conserved amino acid residues among all the ases were chosen to control for the codon sequence and the position effect .
the exon  <dig> ases were then divided into  <dig> groups according to the proximity to exon  <dig>  and the number of translationally preferred and unpreferred codons used in these groups for each conserved amino acid residue were counted .
the frequencies of unpreferred codons indicate the level of constraint due to splicing regulation.
a distance effect  was detected in  <dig> amino acid residues .
a cline in base composition  along the exon cluster could explain the observed distance effect on the synonymous sites in these two codons.
however, there was no correlation between the intron gc-content and the distance from exon  <dig> .
taken together, these two codons in exon  <dig> ases are likely to be among the putative eses that control the distance effect.
although shared ancestry may violate the independence of the analyses, the usage patterns of the same two codons in other drosophila species are largely similar to that in d. melanogaster .
synonymous divergence in the 3' boundary region and the remaining region is similar
the negative correlation observed between codon usage bias and synonymous divergence  <cit>  suggests stronger purifying selection for translationally superior codons in highly biased genes.
if there is purifying selection for ese organization, lower synonymous divergence may be seen in the 3' boundary regions where the effect of splicing regulation is detected.
to investigate this, i calculated synonymous divergence  between different drosophila species in the 3' boundary region and the remaining exonic region of exon  <dig> ases, whose regional differences in cbi appeared to be most apparent .
the synonymous divergences between the two regions using the concatenated sequences of  <dig> orthologous ase pairs were not significantly different in d. melanogaster – d. yakuba, d. yakuba – d. erecta, and d. melanogaster – d. ananassae species-pair comparisons .
similar patterns were also obtained for the constitutive exons of this gene .
although the independence of these tests are violated by overlapping branches shared by these species-pairs, the data indicate that there is no apparent reduction of synonymous divergence due to purifying selection for splicing regulation.
the known eses in human and other species show degenerative features  <cit> , which could be the reason for not observing strong purifying selection for particular codons involved in eses.
however, parmley et al.
<cit>  showed that synonymous sites in putative ese hexamers evolve more slowly than the remaining exonic sequences in mammalian genes.
although i have not specifically examined the difference between putative ese sequences and other exonic sequences, the seeming inconsistency between mammalians and drosophila may arise from the fact that there is no apparent codon bias for translational efficiency in the former.
in drosophila, it is the balance between the intensity of selection for translational efficiency and that for splicing efficiency that determines the relative rate of synonymous site substitutions.
therefore, the absence of apparent reduction of synonymous divergence in the 3' boundary regions compared to the remaining exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that for translational selection, which is known to be nearly neutral  <cit> .
generality of the effect of exonic splicing regulation on synonymous codon usage
the genome-wide phenomenon of reduction of codon bias towards translationally superior codons in intron-exon boundary regions  <cit>  was detected within the ases of exon  <dig> and  <dig> in d. melanogaster.
the strong reduction in the 3' boundary regions in these exons indicates the presence of exonic signals involved in splicing regulation in these regions.
i also examined this feature in other drosophila species with similar codon bias patterns .
the codon usage patterns of the  <dig> drosophila species sequenced to date are similar to those of d. melanogaster, except for d. willistoni which has different usage patterns in some codons  <cit> .
since the codon abundance patterns in 5' and 3' ends of internal exons are highly correlated even between mouse and drosophila  <cit> , it can be assumed that the splice optimum codons are also mostly similar in these species.
the tables in additional files  <dig> and  <dig> indicate significant reductions in most of the species of cbi in the 3' boundary regions of exon  <dig> and  <dig> ases, respectively.
again, the shared ancestry violates independence, these data suggest that selection for splicing efficiency in the 3' regions may be a prominent factor affecting codon usage in these ases.
is it possible that the reduction of the 3' boundary region compared to the remaining 5' region in exon  <dig> and  <dig> ases is due to the relaxation of purifying selection for translational optimum codons per se?
an effect of the relaxed purifying selection is not seen in the synonymous divergence data .
it is intuitively difficult to imagine a particular reason for relaxed selection for translational speed in partial exonic regions.
moreover, selection for translational accuracy favors translation with lower misincorporation rates of codons for functionally important amino acid residues, and thus, conserved amino acid residues should be more highly biased than others  <cit> .
in my data, although the amino acid sequences are conserved in the 3' boundary region  <cit> , the cbi values are reduced.
this observation that the cbi values are reduced in the relatively conserved sequence regions is not consistent with the pattern expected by relaxed translational selection per se in the 3' boundary regions of these dscam ases.
another sensitive comparison using paralogous exonic regions to delineate the effect of splicing regulation on the coding exons was performed.
processed genes that have retrotransposed recently are copies of their parental genes without introns.
these pairs of processed and parental genes enable comparisons between splice-present and splice-absent exonic regions with similar amino acid sequences.
betran et al.
<cit>  have listed  <dig> young retroposed genes and their parental genes  found in the genome of d. melanogaster.
<dig> internal coding exons from the parental genes that were longer than  <dig> bp  were subjected to the following analyses .
cbi in the boundary regions were plotted against those in the central regions , to determine whether cbi in the intron-exon boundary regions are reduced in the parental genes.
in the parental genes, the differences are significant between the center and the 3' boundary  and between center and 5' boundary .
this indicates that most of the intron-exon boundary regions in these internal exons have reduced cbi values, as shown in the whole genome analyses  <cit> .
next, the corresponding paralogous regions in the processed genes were examined.
in the processed genes, the difference between the corresponding regions of the center and 3' boundary, and between the center and 5' boundary, were both not significant .
this means that after intronless retrotransposed copies land onto different genomic locations, the differences between what were formerly the central regions and the boundary regions becomes ambiguous.
this leveling off should be proportional to the time since retroposition.
the degree of leveling off was assessed by plotting the difference in Δ-cbicentral-boundary between the parental and processed genes  against ks of the whole gene  in figure 5c.
a significant relationship between these two values was detected , indicating leveling off of codon usage after the constraint has been removed.
previously, parmley et al.
<cit>  have employed intronless retrogenes in mammals to examine the loss of selective constraints near intron-exon junctions.
they observed higher rates of amino acid evolution near the domains where the intron-exon junctions previously resided, which indicates relaxation of constraints that existed in those boundary regions.
the comparison with the parental genes allows delineation of the course of leveling off of differences in codon bias over time in the processed genes.
this leveling off is a direct prediction of the trade-off model that invokes conflict between translational superiority and splicing regulation near the intron-exon boundary regions  <cit> .
thus, these results corroborate the generality of the effect of splicing regulation on synonymous codon usage which is observed among genome-wide genes in drosophila  <cit> , and supports the conclusions obtained from ases of dscam.
CONCLUSIONS
in the conventional framework of codon usage bias towards translationally superior codons in drosophila, i was able to detect a counteracting selective force of splicing regulation in the 3' intron-exon boundary regions of the dscam exon  <dig> and  <dig> ases.
the positive correlations detected between the expression level  and Δ-cbirest – 3' boundary are clear evidence that synonymous codon usage is restricted by exonic splicing signals in these ases.
this is the first study that directly compare splicing frequency with codon usage.
furthermore, i have shown that two of the codons in exon  <dig> ases may play a role in controlling the distance effect between splice sites, possibly through changing the strength of potential eses by altering codon usage.
a similar synonymous divergence level between different exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that of translational selection.
taken together, these results demonstrate that dscam provides one of the best-controlled and thus sensitive systems to study the effects of such splice-related selection.
in addition, the analyses on the processed genes and their parental genes delineate the generality of the effect of splicing regulation on synonymous codon usage, which is another concrete example of weak natural selection.

 genomic data from many different organisms indicate that synonymous codon usage is typically biased. this bias is an intriguing property that reflects a composite of different evolutionary forces such as mutational bias, genetic drift, and natural selection. in many species, including drosophila, one of the dominant forces affecting the bias is thought to be natural selection for translational efficiency or accuracy, or both. the most abundant codons are preferred because of the abundance of the cognate trnas, and thus the use of the codons maximizes the translation speed and minimizes the amino acid misincorporation rate. there is limited experimental evidence that major codons are translationally superior, but empirical data can be explained well in this framework. the selective force for translational efficiency is weak enough to allow confounding neutral processes to dominate in some cases. for example, the effect of genetic drift is inferred from the comparison between d. melanogaster and d. simulans. other types of selective forces may also counteract translational selection at synonymous sites. splicing regulation is one of them. specifically, there are binding sites within exonic regions for serine-arginine-rich proteins, which enable correct splicing. these binding sites, known as exonic splicing enhancers, are usually located within the vicinity of potential intron-exon junctions, and many of them are ~6– bp in length. these splice signals within the coding regions can potentially affect synonymous codon usage. recent genomic scale analyses in d. melanogaster revealed that translationally optimum codons are not preferred near the intron-exon boundary, and thus are not splice optima. moreover, warnecke and hurst showed that codons putatively involved in eses are almost never translationally optimal, and therefore, a conflict exists between translational advantage and splice efficiency in codon usage. they also showed that this conflict is larger in highly expressed genes, although the effect of expression level was not very strong. these conclusions obtained from a large genomic scale dataset stimulated me to find a well-controlled gene system in which splicing regulation can be effectively analyzed. alternatively spliced exons of down syndrome cell adhesion molecule appeared to be an excellent model system to study such regulation, because this molecule contains the largest number of ases known to date. there are four exons that have multiple alternatively spliced forms, which produces distinct potential isoforms. the structure of the gene is shown in figure these ases can generate unique cell identity by expressing distinct sets of isoforms in the nervous systems where the molecule plays a crucial role in neuronal wiring. also, the large spectrum of distinct isoforms is required in hemocytes where the molecule is necessary for effective immune response. thus, this molecule's unique gene structure has probably arisen from the necessity to generate a diverse repertoire of alternatively spliced variants. in addition to the fact that dscam contains the largest number of ases known to date, these ases provide an ideal system for analyzing the effect of splicing regulation for the following reasons. first of all, these ases are spliced out from a single pre-mrna, and therefore, splicing frequency can be separated from transcription frequency. this kind of within-transcript comparison is the only way to analyze the effect of splicing frequency. these ases are also under the control of the same splicing machinery within the same cell environment in which they are expressed. second, since these ases are in the same genomic region, they are controlled for many possible confounding factors affecting synonymous codon usage such as local gc content and recombination rate. finally, these ases are similar in length and amino acid composition, which are also factors known to influence the codon usage. it should be noted that most of these paralogous ases diverged prior to the d. melanogaster – d. virilis species split, which indicates that the synonymous sites of these ases have diverged beyond saturation. therefore, it is reasonable to assume that their codon usage is effectively independent from each other. additionally, all the species of drosophila whose genomic sequences are available have diverged after the d. melanogaster – d. virilis species split, and thus, orthologous pairs of ases can be identified in many cases. in this study, i analyzed these ases to delineate the effect of splicing regulation on synonymous codon usage by comparing the levels of codon bias in the central and intron-exon boundary regions of these exons. i studied the effect of two splice-related factors that affect the regional differences in cbi among these ases; the splicing frequency and the distance from adjacent constitutive exons. in addition, i also conducted a comparison between paralogous exonic regions of retrotransposed processed genes and their parental genes, in order to investigate the effect of splicing regulation on synonymous codon usage in different gene systems.  codon usage bias towards translationally superior codons is reduced near the 3' intron-exon boundaries in most of the dscam exons genome-wide analysis in drosophila has indicated reduced codon usage bias toward translationally optimal codons within nucleotides of an intron-exon boundary, where the vast majority of functional eses are assumed to exist. in order to investigate whether this trend exists in the ases of dscam exons and i calculated codon bias index in the center and the boundary regions and compared the differences. following warnecke and hurst, the boundary region was defined as the exonic region within full codons of the boundary, excluding the partial codons at the junction, and the center region was defined as the regions remaining after subtracting the flanks. comparisons of cbi values between the center and the boundary regions of these exon clusters are shown in figure cbi values of the 3' boundary region are lower than the central exonic regions in the ases of exons and. among the exon ases, there are significant differences between the center and 3' boundary and 5' boundary and 3' boundary, but not between center and 5' boundary. among the exon ases, the difference between center and 3' boundary is significant, but the differences between center and 5' boundary, and 5' boundary and 3' boundary are not significant. other internal exons which are longer than bp also show significant differences between the center and 3' boundary, the center and 5' boundary, and 5' boundary and 3' boundary. these comparisons indicate that in most of the exons in this gene, codons in the 3' boundary regions are less adapted for translation efficiency compared to codons in the central exonic regions. since the regions analyzed were short, the reduction of cbi in the 3' boundary region could be due to bias in amino acid composition. i compared the codon usage pattern of each amino acid in the 3' boundary region and the remaining 5' region in a pooled sample of exon and ases. among amino acids with degenerate codons, showed higher frequencies of translationally preferred codons in the remaining region compared to those in the 3' boundary region. among them, amino acids showed statistical significance at the level of p < by fisher's exact test. these results clearly indicate that the bias in amino acid composition is not the reason for the reduced cbi in the 3' boundary regions. since warnecke and hurst indicated that translationally optimal codons and splice optimal codons in d. melanogaster are mostly mutually exclusive, my data suggest stronger restriction on codon usage due to splicing regulation in the 3' boundary regions than in the 5' boundary regions throughout the gene, except in the ases of the exon cluster. despite the genome-wide tendency of reduced cbi in the intron-exon boundary regions, various patterns among different genes and exons can arise at least partly by different machineries used for splicing regulation. for example, olson et al. have shown that the heterogeneous nuclear ribosomal protein hrp plays a key role as a splicing repressor in mutually exclusive splicing of the dscam exon ases, but depletion of this protein had no effect on the splicing of ases in other exon clusters. a possible reason for the distinct codon usage pattern in the 3' boundary regions could be that some of the splicing machineries used in this gene utilize signals in these regions to a larger extent than those in other exonic regions. specifically, the reduced cbi3' boundary regionin the ases of the exon cluster, where the difference in cbi between the center and the 3' boundary regions is most apparent, may be explained by the unique model for the mechanism of mutually exclusive alternative splicing proposed by graveley. he discovered two classes of conserved elements in the non-coding regions of the dscam exon cluster; the docking site, located in the intron downstream of constitutive exon and the selector sequences, which are located upstream of each exon ase. each selector sequence is complementary to a portion of the docking site. the model suggests that the formation of the rna structure by docking site:selector sequence interaction is a central component of the mechanism guaranteeing that only one exon ase is included in each dscam mrna. this docking site:selector site interaction brings together the 3' end of exon and the 5' end of one of the exon ases. if this is the case, there must be another factor that brings together the 3' end of the chosen ase and the 5' end of exon the upstream splice reaction aided by docking site:selector site interaction and the downstream reaction may occur simultaneously, but the latter may be where eses are mainly involved. if so, then this could be the reason for observing stronger restriction on the codon usage in the 3' boundary regions of these exon ases. although obtaining a detailed picture of the splicing machineries requires further experiments, the reduction of cbi in the 3' intron-exon boundary regions in this gene suggests that splice-related selection is present. i further examined this possibility by comparing the level of cbi reduction and experimentally obtained splicing frequency data. conflict between translational selection and selection for splice efficiency increases with the splicing frequency iida and akashi have shown that within alternatively spliced genes, gc-ending codons are more abundant in constitutive than in alternatively spliced exons in drosophila and humans. this is consistent with the prediction that translational selection should act more strongly to bias codon usage in constitutive than in alternative exon codons because the former are translated more often than the latter. the selection coefficient for translational selection is expected to become larger in the more frequently translated codons. this is also the pattern that was observed in dscam when whole exonic regions of constitutive and alternative exons were compared; average cbi of internal constitutive exons without utrs was ±, whereas that of ases was ±. as suggested from the comparison of cbi between constitutive and alternative exonic regions, if majority of the dscam exonic region is under translational selection codon usage in the 3' boundary regions and the remaining exonic regions should show different trends in relation to the expression level, because of the additional constraint due to splicing regulation in the former. if the latter is relatively free from the constraint due to splicing regulation, its cbi should show a positive correlation with the expression level due to translational selection. this can be the background level of translational selection. whereas, if synonymous sites in the 3' boundary region are under selection for splicing efficiency, it is expected that cbi in this region should show relatively constant values or a negative correlation with the splicing frequency. the level of conflict, which can be evaluated by the difference between the 3' boundary region and the rest of the region should then increase with the splicing frequency. the expression levels of multiple dscam splice variants have been studied extensively by neves et al. using their custom microarray. taking advantage of the availability of their data, i analyzed the 3' boundary region of the ases in exons and where cbi was significantly reduced in comparison to the remaining 5' region. i used the relative order of the expression level of each exon and ase in hemocyte-derived s cell lines as the estimated order of splicing frequency. in the case of exon most ases are expressed at some or all stages of development, and their expression levels showed only moderate differences among different tissues and among different developmental stages. thus, i used all the ases with the expression level data for the following analyses. as expected, a positive correlation was detected between the expression level and Δ-cbirest – 3' boundary = cbi5' boundary + central – cbi3' boundary, and also between the expression level and the cbi of the remaining 5' region. the correlation between the cbi3' boundary and the splicing frequency was not significant, suggesting a relatively constant level of cbi values in this region with a weak trend of negative correlation with the splicing frequency. the contrasting expression-level dependency of codon usage between the 3' boundary and the remaining exonic regions indicates regional differences in the type of selection affecting the synonymous codon usage within exons of these exon ases. in particular, the positive correlation between Δ-cbirest – 3' boundary and the expression level indicates that the intensity of splice-related selection counteracting the translational selection increases with the splicing frequency. therefore, the conflict between these two evolutionary forces in the 3' boundary region becomes more pronounced in the highly expressed ases of the exon cluster. in the case of exon ases, or of the ases are spliced more frequently than others in a number of tissues. about half of the ases are represented at less than the overrepresentation level of in these tissues. since the expression level order of these less abundant ases is likely to be unreliable, i used only the last half of the ases ordered from the lowest to the highest expression level, where the actual trend in transcript abundance is visible from the heatmap. using these ases of exon a positive correlation between the expression level and Δ-cbirest – 3' boundary was detected. there was no significant correlation between the expression level and cbi of the 3' boundary region or between the expression level and cbi of the remaining 5' region. these results indicate that even with a smaller number of ases analyzed compared to the exon cluster, the intensity of conflict in the exon ases between selection for translational efficiency and for splicing efficiency also correlates with the expression level. the distance to the adjacent constitutive exon affects codon usage in the intron-exon boundary region among the mutually exclusive ases of this gene, the nucleotide distance to the adjacent constitutive exons is a factor, which is likely to affect the occurrence of splicing. presumably, closer splice sites are more likely to interact. this distance effect in ases is analogous to the effect of intron length in the constitutive exons in humans and mouse, in which exons flanked by longer introns contained a significantly higher abundance of putative eses than those flanked by shorter introns. this is consistent with the "exon definition" model of splicing that invokes ese oriented initial splice-site recognition. this model applies better to genes with small exons and large introns, which fits the case of exon and ases. the exon ases spread over a ~ kb region and the distance between splice sites varies between ~ bp to ~ kb. despite this wide distance range, there was no apparent relationship between the position of ases relative to adjacent constitutive exons and the expression level, which suggest that the distance effect is controlled in some way, possibly by exonic signals. it can be inferred from the docking site:selector site model that the downstream splice reaction could be the major function of the eses in the exon ases. if so, the intensity of these splice signals should increase in the ases closer to the upstream adjacent exon to complement the distance effect. inconsistent with this prediction, there was no significant positive correlation between cbi3' boundary and the sequence distance from exon. however, since the intensity of splicing regulation on synonymous codon usage is likely to vary with the position of amino acid residues, specific codons were examined to see if there are variations in codon usage that may contribute to complementing the distance effect. four conserved amino acid residues among all the ases were chosen to control for the codon sequence and the position effect. the exon ases were then divided into groups according to the proximity to exon and the number of translationally preferred and unpreferred codons used in these groups for each conserved amino acid residue were counted. the frequencies of unpreferred codons indicate the level of constraint due to splicing regulation. a distance effect was detected in amino acid residues. a cline in base composition along the exon cluster could explain the observed distance effect on the synonymous sites in these two codons. however, there was no correlation between the intron gc-content and the distance from exon. taken together, these two codons in exon ases are likely to be among the putative eses that control the distance effect. although shared ancestry may violate the independence of the analyses, the usage patterns of the same two codons in other drosophila species are largely similar to that in d. melanogaster. synonymous divergence in the 3' boundary region and the remaining region is similar the negative correlation observed between codon usage bias and synonymous divergence suggests stronger purifying selection for translationally superior codons in highly biased genes. if there is purifying selection for ese organization, lower synonymous divergence may be seen in the 3' boundary regions where the effect of splicing regulation is detected. to investigate this, i calculated synonymous divergence between different drosophila species in the 3' boundary region and the remaining exonic region of exon ases, whose regional differences in cbi appeared to be most apparent. the synonymous divergences between the two regions using the concatenated sequences of orthologous ase pairs were not significantly different in d. melanogaster – d. yakuba, d. yakuba – d. erecta, and d. melanogaster – d. ananassae species-pair comparisons. similar patterns were also obtained for the constitutive exons of this gene. although the independence of these tests are violated by overlapping branches shared by these species-pairs, the data indicate that there is no apparent reduction of synonymous divergence due to purifying selection for splicing regulation. the known eses in human and other species show degenerative features, which could be the reason for not observing strong purifying selection for particular codons involved in eses. however, parmley et al. showed that synonymous sites in putative ese hexamers evolve more slowly than the remaining exonic sequences in mammalian genes. although i have not specifically examined the difference between putative ese sequences and other exonic sequences, the seeming inconsistency between mammalians and drosophila may arise from the fact that there is no apparent codon bias for translational efficiency in the former. in drosophila, it is the balance between the intensity of selection for translational efficiency and that for splicing efficiency that determines the relative rate of synonymous site substitutions. therefore, the absence of apparent reduction of synonymous divergence in the 3' boundary regions compared to the remaining exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that for translational selection, which is known to be nearly neutral. generality of the effect of exonic splicing regulation on synonymous codon usage the genome-wide phenomenon of reduction of codon bias towards translationally superior codons in intron-exon boundary regions was detected within the ases of exon and in d. melanogaster. the strong reduction in the 3' boundary regions in these exons indicates the presence of exonic signals involved in splicing regulation in these regions. i also examined this feature in other drosophila species with similar codon bias patterns. the codon usage patterns of the drosophila species sequenced to date are similar to those of d. melanogaster, except for d. willistoni which has different usage patterns in some codons. since the codon abundance patterns in 5' and 3' ends of internal exons are highly correlated even between mouse and drosophila, it can be assumed that the splice optimum codons are also mostly similar in these species. the tables in additional files and indicate significant reductions in most of the species of cbi in the 3' boundary regions of exon and ases, respectively. again, the shared ancestry violates independence, these data suggest that selection for splicing efficiency in the 3' regions may be a prominent factor affecting codon usage in these ases. is it possible that the reduction of the 3' boundary region compared to the remaining 5' region in exon and ases is due to the relaxation of purifying selection for translational optimum codons per se? an effect of the relaxed purifying selection is not seen in the synonymous divergence data. it is intuitively difficult to imagine a particular reason for relaxed selection for translational speed in partial exonic regions. moreover, selection for translational accuracy favors translation with lower misincorporation rates of codons for functionally important amino acid residues, and thus, conserved amino acid residues should be more highly biased than others. in my data, although the amino acid sequences are conserved in the 3' boundary region, the cbi values are reduced. this observation that the cbi values are reduced in the relatively conserved sequence regions is not consistent with the pattern expected by relaxed translational selection per se in the 3' boundary regions of these dscam ases. another sensitive comparison using paralogous exonic regions to delineate the effect of splicing regulation on the coding exons was performed. processed genes that have retrotransposed recently are copies of their parental genes without introns. these pairs of processed and parental genes enable comparisons between splice-present and splice-absent exonic regions with similar amino acid sequences. betran et al. have listed young retroposed genes and their parental genes found in the genome of d. melanogaster. internal coding exons from the parental genes that were longer than bp were subjected to the following analyses. cbi in the boundary regions were plotted against those in the central regions, to determine whether cbi in the intron-exon boundary regions are reduced in the parental genes. in the parental genes, the differences are significant between the center and the 3' boundary and between center and 5' boundary. this indicates that most of the intron-exon boundary regions in these internal exons have reduced cbi values, as shown in the whole genome analyses. next, the corresponding paralogous regions in the processed genes were examined. in the processed genes, the difference between the corresponding regions of the center and 3' boundary, and between the center and 5' boundary, were both not significant. this means that after intronless retrotransposed copies land onto different genomic locations, the differences between what were formerly the central regions and the boundary regions becomes ambiguous. this leveling off should be proportional to the time since retroposition. the degree of leveling off was assessed by plotting the difference in Δ-cbicentral-boundary between the parental and processed genes against ks of the whole gene in figure 5c. a significant relationship between these two values was detected, indicating leveling off of codon usage after the constraint has been removed. previously, parmley et al. have employed intronless retrogenes in mammals to examine the loss of selective constraints near intron-exon junctions. they observed higher rates of amino acid evolution near the domains where the intron-exon junctions previously resided, which indicates relaxation of constraints that existed in those boundary regions. the comparison with the parental genes allows delineation of the course of leveling off of differences in codon bias over time in the processed genes. this leveling off is a direct prediction of the trade-off model that invokes conflict between translational superiority and splicing regulation near the intron-exon boundary regions. thus, these results corroborate the generality of the effect of splicing regulation on synonymous codon usage which is observed among genome-wide genes in drosophila, and supports the conclusions obtained from ases of dscam.  in the conventional framework of codon usage bias towards translationally superior codons in drosophila, i was able to detect a counteracting selective force of splicing regulation in the 3' intron-exon boundary regions of the dscam exon and ases. the positive correlations detected between the expression level and Δ-cbirest – 3' boundary are clear evidence that synonymous codon usage is restricted by exonic splicing signals in these ases. this is the first study that directly compare splicing frequency with codon usage. furthermore, i have shown that two of the codons in exon ases may play a role in controlling the distance effect between splice sites, possibly through changing the strength of potential eses by altering codon usage. a similar synonymous divergence level between different exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that of translational selection. taken together, these results demonstrate that dscam provides one of the best-controlled and thus sensitive systems to study the effects of such splice-related selection. in addition, the analyses on the processed genes and their parental genes delineate the generality of the effect of splicing regulation on synonymous codon usage, which is another concrete example of weak natural selection. 
in drosophila, genomic data indicates that translationally optimal codons and splice optimal codons are mostly mutually exclusive, and adaptation to translational efficiency is reduced in the intron-exon boundary regions where potential exonic splicing enhancers reside. analyses of the multiple ases of dscam allowed direct tests of the effect of splice-related factors on synonymous codon usage and provided clear evidence that synonymous codon usage bias is restricted by exonic splicing signals near the intron-exon boundary. codon bias indices in the 3' boundary regions were reduced compared to the rest of the exonic regions among and ases of exon and clusters, respectively. synonymous divergence levels between the 3' boundary region and the remaining exonic region of exon ases were similar. additionally, another sensitive comparison of paralogous exonic regions in recently retrotransposed processed genes and their parental genes revealed that, in the former, the differences in cbi between what were formerly the central regions and the boundary regions gradually became smaller over time. a similar synonymous divergence level between the different exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that of translational selection. finally, the leveling off of differences in codon bias over time in retrotransposed genes meets the direct prediction of the tradeoff model that invokes conflict between translational superiority and splicing regulation, and strengthens the conclusions obtained from dscam. these regional differences in cbi were affected by splicing frequency and distance from adjacent exons. down syndrome cell adhesion molecule has the largest number of alternatively spliced exons known to date, and the splicing frequency of each ase is accessible from the relative abundance of the transcript. synonymous codon usage is typically biased towards translationally superior codons in many organisms. thus, these ases comprise a unique model system for studying the effect of splicing regulation on synonymous codon usage.
 synonymous codon usage is typically biased towards translationally superior codons in many organisms. in drosophila, genomic data indicates that translationally optimal codons and splice optimal codons are mostly mutually exclusive, and adaptation to translational efficiency is reduced in the intron-exon boundary regions where potential exonic splicing enhancers reside. in contrast to genomic scale analyses on large datasets, a refined study on a well-controlled set of samples can be effective in demonstrating the effects of particular splice-related factors. down syndrome cell adhesion molecule has the largest number of alternatively spliced exons known to date, and the splicing frequency of each ase is accessible from the relative abundance of the transcript. thus, these ases comprise a unique model system for studying the effect of splicing regulation on synonymous codon usage.  codon bias indices in the 3' boundary regions were reduced compared to the rest of the exonic regions among and ases of exon and clusters, respectively. these regional differences in cbi were affected by splicing frequency and distance from adjacent exons. synonymous divergence levels between the 3' boundary region and the remaining exonic region of exon ases were similar. additionally, another sensitive comparison of paralogous exonic regions in recently retrotransposed processed genes and their parental genes revealed that, in the former, the differences in cbi between what were formerly the central regions and the boundary regions gradually became smaller over time.  analyses of the multiple ases of dscam allowed direct tests of the effect of splice-related factors on synonymous codon usage and provided clear evidence that synonymous codon usage bias is restricted by exonic splicing signals near the intron-exon boundary. a similar synonymous divergence level between the different exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that of translational selection. finally, the leveling off of differences in codon bias over time in retrotransposed genes meets the direct prediction of the tradeoff model that invokes conflict between translational superiority and splicing regulation, and strengthens the conclusions obtained from dscam. 