BACKGROUND
ever since eukaryotic genes were discovered to be interrupted by introns, there has been a heated debate about the origin and evolution of introns.
the "introns-early" school believes that introns were present in the last universal common ancestor of pro- and eukaryotes, and that intron loss is responsible for the lack of introns observed in bacteria.
the "introns-late" school, on the other hand, believes that introns have appeared during the evolution of the eukaryotic lineage, and that intron gain is a frequent event in the evolution leading to the gene structures we see today.
this debate continues to generate a huge amount of literature; for a recent review, see rogozin et al.
<cit> .
on this background, it is surprising that the question of intron position distribution in eukaryotic genes has received relatively little attention.
as an exception to this, it has been observed that introns are not uniformly distributed over the entire gene, but tend to be more abundant close to the 5' end.
this locational bias is especially seen for genes with only a single intron.
sakurai et al.
<cit>  found the 5' bias for genes with a single intron in  <dig> out of  <dig> genomes studied .
in the unicellular organisms saccharomyces cerevisiae and plasmodium falciparum, which have relatively intron-poor genomes, there was a marked 5' bias for all introns.
mourier and jeffares  <cit>  found that the 5' bias was only seen in intron-poor genomes of unicellular organisms.
interestingly, they found no 5' bias in plasmodium where sakurai et al.
<cit>  had seen it.
recently, lin and zhang  <cit>  investigated  <dig> complete eukaryotic genomes and reported that 5' bias was found in all of them, including both uni- and multicellular organisms.
they used a different way of testing this than the other two groups: instead of normalizing all intron positions to a number of bins and adding them up before doing statistical tests, they treated each gene as an independent test and recorded its intron positions as 5' biased, 3' biased, or equally distributed.
it is hypothesized that the origin of the 5' bias is related to the mechanism of intron loss: a spliced mrna can be converted to an intron-less cdna by reverse transcription, and if the cdna then recombines with the gene, one or more introns are lost.
since the reverse transcriptase begins from the 3' end of the mrna, incomplete cdnas predominantly represent the 3' end of the gene, and therefore, intron loss preferentially occurs in the 3' end  <cit> .
also intron gain seems to occur preferentially in the 3' end, sverdlov et al.
<cit>  reported.
they found that phylogenetically old introns  showed an excess in the 5' end, while new introns in intron-rich genomes were found preferentially in the 3' end.
the 5' end of an intron is referred to as the donor site, and the 3' end as the acceptor site.
the position of the donor and acceptor sites relative to the reading frame is referred to as the phase of the intron: a phase  <dig> intron is positioned between two codons, while a phase  <dig> intron disrupts a codon after the first position and a phase  <dig> intron after the second position.
in most coding regions, phase  <dig> introns are the most common, followed by phase  <dig> introns and then phase  <dig> introns as the least common  <cit> .
in proteins with secretory signal peptides, however, phase  <dig> introns are the most common  <cit> .
introns are recognised by the spliceosome, a complex of several small ribonucleoprotein particles   <cit> .
there is an almost completely conserved consensus sequence for the donor and acceptor sites, the two first positions in the intron being "gt" and the last two positions "ag".
exceptions to this rule exist, but they are rare.
burset et al.
<cit>  did a comprehensive analysis of est-supported canonical and non-canonical splice sites and reported that  <dig> % of the introns, after correcting errors, had sequences other than "gt...ag."
the majority of these  were "gc...ag," and the other patterns each comprised less than  <dig> %.
it has been found that introns with the non-canonical sequence "at...ac" comprise a special group recognised by its own spliceosome, where the rare u <dig> and u <dig> snrnps have replaced the standard u <dig> and u <dig> particles  <cit> .
there is also some weaker sequence conservation on the exon side of donor and acceptor sites.
the consensus sequence for the exon-exon junction is "ag|g" where the "|" denotes the position of the intron  <cit> .
this has been described as the "proto-splice site" as it is assumed that new introns are predominantly inserted into an "ag|g" site  <cit> .
coghlan and wolfe  <cit>  indeed found that recently gained introns  had a stronger "ag|g" consensus than older introns.
however, the "introns-early" school, who tends to be skeptical of the notion of intron gain, has found that the occurrence of "proto-splice sites" does not agree with the distribution of intron phases found in extant organisms  <cit> .
the existence of a nucleotide consensus flanking existing introns is, of course, no guarantee that the sites are remnants of original proto-splice sites; they might also have evolved convergently after the introns appeared in order to adapt to the splicing machinery.
sverdlov et al.
<cit>  addressed this question by examining the context of introns inserted at amino acids that were totally conserved between eight diverse eukaryotic genomes.
by considering only nucleotides that could not be changed without changing the amino acid, they arrived at a splice site sequence context that had not been modified by directional selection, and it turned out to have the same consensus as that of all splice sites.
the conclusion is that either introns have been inserted preferentially into proto-splice sites, or they have been inserted at random but preferentially fixed if the sequence context was a proto-splice site.
another question is whether shared intron positions reflects evolutionary conservation or parallel gain of new introns.
qiu et al.
<cit> , using a bayesian modeling of intron evolution, found that most introns shared between distantly related species are results of parallel gains.
this is in contrast to two more recent papers.
sverdlov et al.
<cit>  constructed a dynamic model of intron insertion by using a weight matrix for the proto-splice sites and inserting introns with a probability proportional to the weight matrix score.
their simulated results suggest that only a small fraction  of shared intron positions in distantly related species are due to parallel gains.
nguyen et al.
<cit>  used a maximum likelihood estimator where the number of target sites  was treated as an parameter to be estimated, and their results suggest that parallel gains account for ≈ <dig> % of shared intron positions.
when comparing the genes for cytoplasmic ribosomal proteins to those for mitochondrial ribosomal proteins, however, the same group found that all shared intron positions between these two groups resulted from parallel gains  <cit> ; a result consistent with the introns-late view that the bacterial ancestor of mitochondria did not have introns.
it is a matter of debate whether intron position correlates with protein structure.
if the "introns-early" theory, also called the "exon theory of genes" is correct, proteins should have evolved by assembly of small autonomous modules or structural domains, and one should expect that exons in proteins will correspond to boundaries of such modules.
stoltzfus et al.
<cit>  tested this and found no correlation between intron position and protein structure.
on the other hand, de souza et al.
<cit>  found that phase  <dig> introns were indeed found at boundaries of compact protein modules, and fedorov et al.
<cit>  reported that this was especially true for phylogenetically old proteins .
recently, whamond and thornton  <cit>  analysed intron positions in relation to amino acids and protein secondary structure.
they found that the distribution of intron positions in the three structural classes helix, sheet, and coil was different from the background distribution, but this bias could be largely explained by the nucleotide preferences surrounding the introns .
tordai and patthy  <cit>  investigated the distribution of introns in human genes with and without signal peptides, and found a significant excess of phase  <dig> introns in the vicinity of the signal peptide cleavage site.
the reason for this is hypothesized to be that proto-splice sites of phase  <dig> correspond to glycine codons , and positions - <dig>  - <dig>  - <dig> and - <dig> relative to the cleavage site are significantly enriched in glycine.
it also correlates well with the fact that extracellular proteins often have evolutionary modules or domains that are bounded in both ends by phase  <dig> introns  <cit> .
recently, vibranovski et al.
<cit>   reinvestigated this phenomenon and reported that all "g|g" intron contexts, not only "ag|g" proto-splice sites, were enriched in the region of the signal peptide cleavage site, and that the entire sequence of proteins with signal peptides were enriched in phase  <dig> introns.
on this background, they claim that exon shuffling, rather than intron insertion into proto-splice sites, must be the explanation for the phase  <dig> signal peptide peak.
RESULTS
intron length distribution
during splicing, the intron assumes a lariat structure, where the 5' end is covalently attached to a branch point a short distance upstream of the 3' end  <cit> .
the intron must have a certain minimum length for this lariat formation to be possible, but exactly how short an intron can be is a matter of some debate.
goodall and filipowicz  <cit> , using deletion mutants, found that the minimum functional intron length in plants  was between  <dig> and  <dig> nt.
they noted that this length requirement is similar to that seen in vertebrates, but significantly greater than that in fungi and insects.
introns shorter than this occur in genbank.
the shortest of them are probably annotation errors, but where should we set the cutoff?
in order to find a non-arbitrary lower length threshold for introns, the intron length distribution was calculated on the genbank data – vertebrates, arthropods, fungi, and flowering plants .
in the length distribution dataset, introns without the canonical "gt...ag" sequence were not weeded out.
in all organism groups, the second most abundant splice site sequence was "gc...ag", comprising  <dig> – <dig> % of the introns .
this is in agreement with earlier observations  <cit> .
intron length distribution was calculated for introns with non-consensus splice sites separately, and it was found that many of them  were less than  <dig> nucleotides in length.
in fact, these gaps in the coding sequence probably do not represent introns, but programmed translational frameshifts  <cit> .
in figure  <dig>  the cumulative distribution of intron lengths in the four organism groups is shown.
it is apparent that the length distributions are very different for the four groups.
introns shorter than  <dig> nt accounted for  <dig> % of the fungal introns, but only  <dig> % of the vertebrate introns.
the non-cumulated distributions of intron lengths are shown in supplementary figure s <dig> .
the curves in figure  <dig> all have a linear domain.
we defined a minimum length cutoff by drawing a line through the linear domain and extending it to the x-axis.
this yielded the following thresholds: vertebrates:  <dig> nt; arthropods:  <dig> nt; fungi:  <dig> nt; and plants:  <dig> nt.
these values are in agreement with those of goodall and filipowicz  <cit> .
intron position statistics
the distributions of intron positions within the first  <dig> amino acid positions of eukaryotic genes from the genbank sets are shown in figure  <dig>  in the plots of genes without signal peptides , it can be seen that phase  <dig> introns are more frequent than phase  <dig> introns which are again more frequent than phase  <dig> introns, in agreement with what has been described before  <cit> .
the excess of phase  <dig> introns is particularly pronounced in plant genes.
for proteins with signal peptides , there is an excess of phase  <dig> introns in a region around  <dig> for the two animal groups, and between  <dig> and  <dig> in plants.
this has been described as corresponding to the positions of the signal peptide cleavage sites  <cit> , cf.
the background section.
the phase  <dig> excess cannot be seen for the fungal sequences, where proteins with signal peptides and introns are simply too rare to make this type of statistics.
in proteins without signal peptides , another phenomenon is seen: a sharp peak of phase  <dig> introns at position  <dig>  i.e.
between the start codon and codon  <dig>  this is what we refer to as the start codon introns.
note that there are no values for phase  <dig> introns at position  <dig> – this would be introns occurring immediately before the start codon, and those are not visible in the genbank coding sequence  features the data are derived from .
the peak of start codon introns is very high in vertebrates, arthropods and fungi.
in plants, it is not higher than the peaks for phase  <dig> introns between positions  <dig> and  <dig>  but it is still conspicuous against the relatively intron-poor region between positions  <dig> and  <dig>
note that introns disrupting the start codon, i.e.
phase  <dig> and phase  <dig> introns in position  <dig>  are relatively rare, especially in plants.
in the genome data sets, both the phase  <dig> peak for proteins with signal peptides and the start codon peak for proteins without signal peptides are clearly visible .
the intron position distributions for the human genome and the mouse genome look very similar to the vertebrate genbank set, and the drosophila genome shows the same pattern as the arthropod genbank set.
start codon introns have been briefly mentioned in the literature before, but only for human ribosomal proteins.
in a paper about the human ribosomal protein genes, yoshihama et al.
<cit>  remarked: "interestingly, the atg was always located near the splice sites of the first intron and, in  <dig> cases, was exactly at the 3' end of the first exon" .
the phenomenon is not limited to ribosomal proteins, however.
to address this question, we made plots of the genome data sets without signal peptides with ribosomal proteins removed .
these plots are shown in supplementary figure s <dig> .
in the plots, there is only a slight lowering of the start codon peak; the relative frequency being  <dig>  for human,  <dig>  for mouse, and  <dig>  for fly ; still well outside the range of frequencies for other positions.
in arthropod genes with signal peptides, a peak of phase  <dig> introns is seen at position  <dig> .
we checked the genbank annotation of these  <dig> proteins and found that  <dig> of them were from the genome sequence of drosophila melanogaster.
indeed, the same peak can be seen in the non-homology reduced drosophila genome , with the peak containing  <dig> proteins.
a curious observation is that  <dig> of the  <dig> introns and  <dig> of the  <dig> introns  are the only introns in their respective genes; for comparison, only  <dig> % of all the d. melanogaster genes with signal peptides have exactly one intron.
the functional annotation of the  <dig> peak proteins reveals a striking fact:  <dig> of them  are structural constituents of cuticle .
<dig> have functional annotation missing or "unknown," and only  <dig> have an annotation of something other than cuticle.
for comparison, only  <dig> % of all the d. melanogaster genes with signal peptides are annotated with "structural constituent ...
cuticle."
in arthropods, fungi and plants without signal peptides, a slight downward slope can be seen in the intron frequency.
we tested whether this is a real 5' bias by computing the correlation coefficient between position and relative frequency for positions  <dig> through  <dig>  fungi and plants have significant  downward slopes for all three phases, arthropods only for phase  <dig> and phase  <dig>  the vertebrate set shows no slope.
if the proteins are aligned by the stop codon instead of the start codon, the picture shown in figure  <dig> emerges.
there are no stop codon peaks, except for a weak phase  <dig> peak before the last codon in plants without signal peptides.
for proteins with signal peptides, no special features can be seen.
observe that it is only in proteins without signal peptides that phase  <dig> introns are the most abundant.
for proteins without signal peptides, a slight reduction in the intron frequency for all three phases can be seen in the last approximately  <dig> positions .
proteins with start codon introns
in order to characterize the start codon introns, they were compared to a background set.
both the start codon introns and the background set were derived from proteins without signal peptides.
to eliminate possible bias from the fact that start codon introns are always phase  <dig> and always the first intron in the coding sequence ), the background set was limited to phase  <dig> introns that were the first in the coding sequence.
a comparison of intron lengths was inconclusive.
for vertebrates, start codon introns are slightly shorter than the background set, but for fungi they are slightly longer.
for arthropods and plants, there are no significant differences .
when examining the nucleotide distribution, significant differences are found for vertebrates, fungi, and plants , but not for arthropods .
when using nucleotide pair frequencies, significant differences are found for all four groups  .
however, the results show no clear trends – there are no dinucleotide preferences that are the same in all four organism groups.
the sets of proteins without signal peptides carrying a start codon intron were also subjected to further analysis, and here, some interesting differences were found.
again, both the positive and the negative set consisted of proteins without signal peptides.
the first thing we noticed was that proteins with start codon introns are on average shorter than other proteins, see table  <dig>  the differences are significant on the 5% level for vertebrates and fungi, and better than the  <dig> % level for arthropods and plants .
average length  and amino acid frequencies for proteins without signal peptides, divided into proteins with  and without  start codon introns.
where the difference for a particular amino acid is greater than  <dig> %, the higher percentage is shown in boldface.
the amino acid distributions were also calculated, and it was found that there are significant differences between proteins with and without start codon introns for all organism groups .
the amino acid frequencies are shown in table  <dig>  the most conspicuous differences, seen in all four organism groups, are that lysine  is more abundant in proteins with start codon introns than in other proteins, while serine  is less abundant.
in order to assess the conservation of start codon intron proteins, human genome proteins involved in a reciprocal best hit  to mouse or fly were compared.
while human proteins with a start codon intron on average have  <dig> % identities in the global alignments with mouse, the other proteins have only  <dig> % .
for the alignments of human versus drosophila, the proteins with start codon introns had  <dig> % identities, while the others had  <dig> % .
only rbhs where the human gene was predicted not to contain a signal peptide were included in these calculations.
we also tested whether start codon intron positions are more conserved than other intron positions.
all intron positions were mapped onto the global alignments, and in positions where the human protein had an intron it was tested if the mouse or fly protein also had an intron.
in the comparison of human versus fly, there are  <dig> rbhs with a human start codon intron.
<dig> had a corresponding fly phase  <dig> intron, and  <dig> of these  were also in a start codon position.
for comparison, only  <dig> of the  <dig>  human phase  <dig> introns in other positions are conserved in fly.
this difference is highly significant .
in the comparison between human and mouse, where the overall conservation of phase  <dig> intron positions is as high as  <dig> %, no significant difference is seen.
when looking at the functional annotations, a curious observation was made: in the sets of proteins with start codon introns, ribosomal proteins are overrepresented.
this, of course, corresponds well with the fact that ribosomal proteins was the first place where start codon introns were observed.
in the vertebrates, there are  <dig> ribosomal proteins out of the  <dig> start codon intron proteins, corresponding to 29%, while the background frequency in the non-start codon intron proteins without signal peptides is  <dig> %.
in the plants, there are  <dig> out of  <dig>  against a background frequency of  <dig> %, and in arthropods, there are  <dig> out of  <dig>  against  <dig> %.
in fungi, there are only  <dig> ribosomal proteins out of  <dig> in the start codon intron set, but the overrepresentation is still significant .
the descriptions of all start codon intron proteins from the genbank data sets can be seen in supplementary table s <dig> .
in the genome sets, we found the same phenomenon: among human genes with start codon introns, there are  <dig> % proteins marked "ribosomal" against a background frequency of  <dig> %; in mouse, the frequencies are  <dig> % against  <dig> %, and in fly  <dig> % against  <dig> %.
these differences are highly significant .
since ribosomal proteins can be expected to be more conserved than other proteins, we investigated whether the higher conservation of proteins with start codon introns could be solely due to the high proportion of ribosomal proteins.
the lists of rbhs were constrained to those where the human protein description did not contain the word "ribosomal," and the percent identities were compared again.
the difference is still significant, although smaller .
discussion
when observing the start codon peak of introns in eukaryotic genes, the question naturally arose whether this was a biological phenomenon or some kind of artifact of gene finding.
as mentioned in the description of the genbank data sets, we discarded genes where the evidence was marked as "not experimental."
however, we did not limit the data set to genes marked "/evidence=experimental," as this yielded too few genes to carry out the statistical analysis.
the majority of genes do not have an "/evidence" qualifier at all.
the genbank data may therefore contain genes inferred by prediction.
in the whole-genome data, on the other hand, we discarded the genes solely based on prediction  and here, the start codon peak was still seen.
this strongly suggests that the overabundance of start codon introns is a biological phenomenon.
also, the observation that proteins carrying a start codon intron comprise a special class points in this direction.
we have shown that proteins with start codon introns are relatively short, lysine-rich, serine-poor and evolutionarily conserved.
in addition, we have shown that the start codon intron positions are often conserved between human and fly.
during the peer review process it was suggested that our method for showing the excess of phase  <dig> introns after the start codon could be questionable.
the concern was that that the method of aligning the first  <dig> aa of all protein sequences, while showing the start codon peak, might fail to detect some other peaks.
the reason is that insertions and deletions during evolution would alter the distance between start codon and intron peak, thereby spreading the peak over several positions.
in other words, peaks in other positions have been "diluted" over evolution while the start codon peak is retained simply because we use the start codon as "anchor point" for our alignment.
this "dilution effect" would be enhanced by the fact that the n-terminal parts of proteins are often less conserved than other parts, with many indels.
the reviewer suggested that we specifically analyzed proteins that have their n-terminals conserved over evolution to avoid this suggested effect.
we did this by selecting from the list of rbhs between human and drosophila those global alignments where neither the human nor the fly sequence had gaps within the first  <dig> positions.
this yielded  <dig> pairs, of which  <dig> human sequences and  <dig> fly sequences were predicted to be without signal peptides.
we then repeated the intron position statistics for these sequences, and the result is very clear: the phase  <dig> start codon peaks are even more conspicuous than those in figure  <dig> , and there are no peaks in other positions .
furthermore, the fact that we can still see the phase  <dig> signal peptide peak in our start codon alignments despite the length variation of signal peptides suggests that the "dilution effect" does not completely wipe out peaks relatively close to the n-terminus, but only makes them broader.
the reviewer suggested that the reason could be that proteins with signal peptides may have their n-terminals conserved over evolution, but this is not likely to be the case, since signal peptides are known to evolve rapidly  <cit> .
the function of the start codon intron may be to allow the 5' utr to participate in exon shuffling, so that different genes can exchange regulatory information.
as described in the background section, phase  <dig> introns are often found at the boundaries of evolutionary modules in proteins without signal peptides  <cit> , and this effect is stronger in phylogenetically old proteins  <cit> .
this would correspond quite well with the 5' utr serving as an evolutionary module, separated from the protein coding domain by a phase  <dig> intron at the start codon, especially in well-conserved proteins.
if the start codon intron really is a boundary of an evolutionary module, one should expect that the second intron would often be a phase  <dig> intron, too, since modules in exon shuffling most often have introns of the same phase in both ends  <cit> .
we tried to investigate this by counting the phases of the second introns in proteins where the first intron was a start codon intron, compared with proteins where the first intron was a phase  <dig> intron in another position.
in vertebrates and plants, there was indeed a tendency towards a higher proportion of second phase  <dig> introns for the start codon intron set, but it was not significant.
in arthropods there was no difference, and in fungi it was in fact lower .
another explanation for the start codon introns could be that the nucleotide context of codons  <dig> and  <dig> provide a proto-splice site.
the codon before is necessarily "atg," so the two nucleotides before the intron is "tg" instead of the canonical "ag."
however, the "a" is not very well conserved, and "t" is the next most common nucleotide in the - <dig> position relative to the intron  <cit> .
also, in the evolutionarily conserved amino acids examined by sverdlov et al.
<cit>  in order to reconstruct ancestral proto-splice sites , methionine was slightly overrepresented immediately before a phase  <dig> intron.
consequently, the start codon could be part of a proto-splice site if the first position of codon  <dig> is a "g".
indeed, "g" is enriched in position + <dig>  in eukaryotes  <cit> .
however, this enrichment is much higher in plants than in vertebrates or drosophila  <cit> , so if this were to be the sole explanation for start codon introns, we should expect a much stronger start codon intron signal in plants than in animals, and this is not the case .
on the other hand, the relatively weak start codon intron signal in plants could be explained within the proto-splice site model if there is a much higher frequency of proto-splice sites in the plant genes than in the vertebrate or arthropod genes as a whole.
we have not checked whether this is the case.
the 5' bias we observed in the position of introns within the non-vertebrate genes is in contrast to mourier and jeffares  <cit> , who reported that introns in multicellular genomes are evenly distributed throughout genes.
our fungal data set does contain unicellular organisms, but arthropods and plants do not.
on the other hand, sakurai et al.
and lin and zhang  <cit>  did find a 5' bias in virtually all genomes studied.
interestingly, they even found 5' bias in human and mouse, where we did not observe it.
the fact that ribosomal proteins are strongly overrepresented in start codon intron proteins is striking, because it is an example of an intron position being conserved in a functional class of proteins that are not related by descent .
to be absolutely sure our homology reduction procedure had not let any pairs of homologous sequences through, we performed an all-versus-all global alignment of the sequences with start codon introns in all the four genbank organism groups.
no pairs showed more than  <dig> % identity in the global alignment.
we also made phylogenetic trees of the start codon intron sets and found no clusters .
plots of % identity versus alignment score and the trees can be seen in supplementary figure s <dig>  section  <dig> .
another example of this is the position  <dig> phase  <dig> peak in drosophila.
initially, we thought that it could be a gene finding artifact, since it was specifically seen in one genome.
however, the extremely high proportion of cuticle constituents convinced us that this too is a biological phenomenon.
it is also not entirely unknown in the literature: rondot et al.
<cit> , while describing two larval-pupal cuticle protein genes from the insect tenebrio molitor, found that both had a single intron within the signal peptide coding region .
in addition to their own observation, they remarked that such an intron localization  has been described in cuticle protein genes from many different species, including drosophila.
again, note that this is not simply a gene family with a conserved intron position, since the peak is also seen in the heavily homology-reduced set.
in order to test this further, we did an all-versus-all global alignment of the  <dig> sequences in the arthropod peak and found no pairs with more than  <dig> % identity .
CONCLUSIONS
the start codon peak of phase  <dig> introns is so conspicuous that it is quite remarkable that it has only been described in a single sentence in the literature before.
as mentioned above, yoshihama et al.
<cit>  saw the phenomenon in human ribosomal proteins, but to the best of our knowledge we are the first to provide a general investigation of it.
in this paper, we have filled this gap in the description of intron positions in genes and argued that this phenomenon seems to be a biological reality and not simply an artifact of gene finding or alignment.

 ever since eukaryotic genes were discovered to be interrupted by introns, there has been a heated debate about the origin and evolution of introns. the "introns-early" school believes that introns were present in the last universal common ancestor of pro- and eukaryotes, and that intron loss is responsible for the lack of introns observed in bacteria. the "introns-late" school, on the other hand, believes that introns have appeared during the evolution of the eukaryotic lineage, and that intron gain is a frequent event in the evolution leading to the gene structures we see today. this debate continues to generate a huge amount of literature; for a recent review, see rogozin et al.. on this background, it is surprising that the question of intron position distribution in eukaryotic genes has received relatively little attention. as an exception to this, it has been observed that introns are not uniformly distributed over the entire gene, but tend to be more abundant close to the 5' end. this locational bias is especially seen for genes with only a single intron. sakurai et al. found the 5' bias for genes with a single intron in out of genomes studied. in the unicellular organisms saccharomyces cerevisiae and plasmodium falciparum, which have relatively intron-poor genomes, there was a marked 5' bias for all introns. mourier and jeffares found that the 5' bias was only seen in intron-poor genomes of unicellular organisms. interestingly, they found no 5' bias in plasmodium where sakurai et al. had seen it. recently, lin and zhang investigated complete eukaryotic genomes and reported that 5' bias was found in all of them, including both uni- and multicellular organisms. they used a different way of testing this than the other two groups: instead of normalizing all intron positions to a number of bins and adding them up before doing statistical tests, they treated each gene as an independent test and recorded its intron positions as 5' biased, 3' biased, or equally distributed. it is hypothesized that the origin of the 5' bias is related to the mechanism of intron loss: a spliced mrna can be converted to an intron-less cdna by reverse transcription, and if the cdna then recombines with the gene, one or more introns are lost. since the reverse transcriptase begins from the 3' end of the mrna, incomplete cdnas predominantly represent the 3' end of the gene, and therefore, intron loss preferentially occurs in the 3' end. also intron gain seems to occur preferentially in the 3' end, sverdlov et al. reported. they found that phylogenetically old introns showed an excess in the 5' end, while new introns in intron-rich genomes were found preferentially in the 3' end. the 5' end of an intron is referred to as the donor site, and the 3' end as the acceptor site. the position of the donor and acceptor sites relative to the reading frame is referred to as the phase of the intron: a phase intron is positioned between two codons, while a phase intron disrupts a codon after the first position and a phase intron after the second position. in most coding regions, phase introns are the most common, followed by phase introns and then phase introns as the least common. in proteins with secretory signal peptides, however, phase introns are the most common. introns are recognised by the spliceosome, a complex of several small ribonucleoprotein particles. there is an almost completely conserved consensus sequence for the donor and acceptor sites, the two first positions in the intron being "gt" and the last two positions "ag". exceptions to this rule exist, but they are rare. burset et al. did a comprehensive analysis of est-supported canonical and non-canonical splice sites and reported that % of the introns, after correcting errors, had sequences other than "gt...ag." the majority of these were "gc...ag," and the other patterns each comprised less than %. it has been found that introns with the non-canonical sequence "at...ac" comprise a special group recognised by its own spliceosome, where the rare u and u snrnps have replaced the standard u and u particles. there is also some weaker sequence conservation on the exon side of donor and acceptor sites. the consensus sequence for the exon-exon junction is "ag|g" where the "|" denotes the position of the intron. this has been described as the "proto-splice site" as it is assumed that new introns are predominantly inserted into an "ag|g" site. coghlan and wolfe indeed found that recently gained introns had a stronger "ag|g" consensus than older introns. however, the "introns-early" school, who tends to be skeptical of the notion of intron gain, has found that the occurrence of "proto-splice sites" does not agree with the distribution of intron phases found in extant organisms. the existence of a nucleotide consensus flanking existing introns is, of course, no guarantee that the sites are remnants of original proto-splice sites; they might also have evolved convergently after the introns appeared in order to adapt to the splicing machinery. sverdlov et al. addressed this question by examining the context of introns inserted at amino acids that were totally conserved between eight diverse eukaryotic genomes. by considering only nucleotides that could not be changed without changing the amino acid, they arrived at a splice site sequence context that had not been modified by directional selection, and it turned out to have the same consensus as that of all splice sites. the conclusion is that either introns have been inserted preferentially into proto-splice sites, or they have been inserted at random but preferentially fixed if the sequence context was a proto-splice site. another question is whether shared intron positions reflects evolutionary conservation or parallel gain of new introns. qiu et al., using a bayesian modeling of intron evolution, found that most introns shared between distantly related species are results of parallel gains. this is in contrast to two more recent papers. sverdlov et al. constructed a dynamic model of intron insertion by using a weight matrix for the proto-splice sites and inserting introns with a probability proportional to the weight matrix score. their simulated results suggest that only a small fraction of shared intron positions in distantly related species are due to parallel gains. nguyen et al. used a maximum likelihood estimator where the number of target sites was treated as an parameter to be estimated, and their results suggest that parallel gains account for ≈ % of shared intron positions. when comparing the genes for cytoplasmic ribosomal proteins to those for mitochondrial ribosomal proteins, however, the same group found that all shared intron positions between these two groups resulted from parallel gains; a result consistent with the introns-late view that the bacterial ancestor of mitochondria did not have introns. it is a matter of debate whether intron position correlates with protein structure. if the "introns-early" theory, also called the "exon theory of genes" is correct, proteins should have evolved by assembly of small autonomous modules or structural domains, and one should expect that exons in proteins will correspond to boundaries of such modules. stoltzfus et al. tested this and found no correlation between intron position and protein structure. on the other hand, de souza et al. found that phase introns were indeed found at boundaries of compact protein modules, and fedorov et al. reported that this was especially true for phylogenetically old proteins. recently, whamond and thornton analysed intron positions in relation to amino acids and protein secondary structure. they found that the distribution of intron positions in the three structural classes helix, sheet, and coil was different from the background distribution, but this bias could be largely explained by the nucleotide preferences surrounding the introns. tordai and patthy investigated the distribution of introns in human genes with and without signal peptides, and found a significant excess of phase introns in the vicinity of the signal peptide cleavage site. the reason for this is hypothesized to be that proto-splice sites of phase correspond to glycine codons, and positions - - - and - relative to the cleavage site are significantly enriched in glycine. it also correlates well with the fact that extracellular proteins often have evolutionary modules or domains that are bounded in both ends by phase introns. recently, vibranovski et al. reinvestigated this phenomenon and reported that all "g|g" intron contexts, not only "ag|g" proto-splice sites, were enriched in the region of the signal peptide cleavage site, and that the entire sequence of proteins with signal peptides were enriched in phase introns. on this background, they claim that exon shuffling, rather than intron insertion into proto-splice sites, must be the explanation for the phase signal peptide peak.  intron length distribution during splicing, the intron assumes a lariat structure, where the 5' end is covalently attached to a branch point a short distance upstream of the 3' end. the intron must have a certain minimum length for this lariat formation to be possible, but exactly how short an intron can be is a matter of some debate. goodall and filipowicz, using deletion mutants, found that the minimum functional intron length in plants was between and nt. they noted that this length requirement is similar to that seen in vertebrates, but significantly greater than that in fungi and insects. introns shorter than this occur in genbank. the shortest of them are probably annotation errors, but where should we set the cutoff? in order to find a non-arbitrary lower length threshold for introns, the intron length distribution was calculated on the genbank data – vertebrates, arthropods, fungi, and flowering plants. in the length distribution dataset, introns without the canonical "gt...ag" sequence were not weeded out. in all organism groups, the second most abundant splice site sequence was "gc...ag", comprising – % of the introns. this is in agreement with earlier observations. intron length distribution was calculated for introns with non-consensus splice sites separately, and it was found that many of them were less than nucleotides in length. in fact, these gaps in the coding sequence probably do not represent introns, but programmed translational frameshifts. in figure the cumulative distribution of intron lengths in the four organism groups is shown. it is apparent that the length distributions are very different for the four groups. introns shorter than nt accounted for % of the fungal introns, but only % of the vertebrate introns. the non-cumulated distributions of intron lengths are shown in supplementary figure s. the curves in figure all have a linear domain. we defined a minimum length cutoff by drawing a line through the linear domain and extending it to the x-axis. this yielded the following thresholds: vertebrates: nt; arthropods: nt; fungi: nt; and plants: nt. these values are in agreement with those of goodall and filipowicz. intron position statistics the distributions of intron positions within the first amino acid positions of eukaryotic genes from the genbank sets are shown in figure in the plots of genes without signal peptides, it can be seen that phase introns are more frequent than phase introns which are again more frequent than phase introns, in agreement with what has been described before. the excess of phase introns is particularly pronounced in plant genes. for proteins with signal peptides, there is an excess of phase introns in a region around for the two animal groups, and between and in plants. this has been described as corresponding to the positions of the signal peptide cleavage sites, cf. the background section. the phase excess cannot be seen for the fungal sequences, where proteins with signal peptides and introns are simply too rare to make this type of statistics. in proteins without signal peptides, another phenomenon is seen: a sharp peak of phase introns at position i.e. between the start codon and codon this is what we refer to as the start codon introns. note that there are no values for phase introns at position – this would be introns occurring immediately before the start codon, and those are not visible in the genbank coding sequence features the data are derived from. the peak of start codon introns is very high in vertebrates, arthropods and fungi. in plants, it is not higher than the peaks for phase introns between positions and but it is still conspicuous against the relatively intron-poor region between positions and note that introns disrupting the start codon, i.e. phase and phase introns in position are relatively rare, especially in plants. in the genome data sets, both the phase peak for proteins with signal peptides and the start codon peak for proteins without signal peptides are clearly visible. the intron position distributions for the human genome and the mouse genome look very similar to the vertebrate genbank set, and the drosophila genome shows the same pattern as the arthropod genbank set. start codon introns have been briefly mentioned in the literature before, but only for human ribosomal proteins. in a paper about the human ribosomal protein genes, yoshihama et al. remarked: "interestingly, the atg was always located near the splice sites of the first intron and, in cases, was exactly at the 3' end of the first exon". the phenomenon is not limited to ribosomal proteins, however. to address this question, we made plots of the genome data sets without signal peptides with ribosomal proteins removed. these plots are shown in supplementary figure s. in the plots, there is only a slight lowering of the start codon peak; the relative frequency being for human, for mouse, and for fly; still well outside the range of frequencies for other positions. in arthropod genes with signal peptides, a peak of phase introns is seen at position. we checked the genbank annotation of these proteins and found that of them were from the genome sequence of drosophila melanogaster. indeed, the same peak can be seen in the non-homology reduced drosophila genome, with the peak containing proteins. a curious observation is that of the introns and of the introns are the only introns in their respective genes; for comparison, only % of all the d. melanogaster genes with signal peptides have exactly one intron. the functional annotation of the peak proteins reveals a striking fact: of them are structural constituents of cuticle. have functional annotation missing or "unknown," and only have an annotation of something other than cuticle. for comparison, only % of all the d. melanogaster genes with signal peptides are annotated with "structural constituent... cuticle." in arthropods, fungi and plants without signal peptides, a slight downward slope can be seen in the intron frequency. we tested whether this is a real 5' bias by computing the correlation coefficient between position and relative frequency for positions through fungi and plants have significant downward slopes for all three phases, arthropods only for phase and phase the vertebrate set shows no slope. if the proteins are aligned by the stop codon instead of the start codon, the picture shown in figure emerges. there are no stop codon peaks, except for a weak phase peak before the last codon in plants without signal peptides. for proteins with signal peptides, no special features can be seen. observe that it is only in proteins without signal peptides that phase introns are the most abundant. for proteins without signal peptides, a slight reduction in the intron frequency for all three phases can be seen in the last approximately positions. proteins with start codon introns in order to characterize the start codon introns, they were compared to a background set. both the start codon introns and the background set were derived from proteins without signal peptides. to eliminate possible bias from the fact that start codon introns are always phase and always the first intron in the coding sequence ), the background set was limited to phase introns that were the first in the coding sequence. a comparison of intron lengths was inconclusive. for vertebrates, start codon introns are slightly shorter than the background set, but for fungi they are slightly longer. for arthropods and plants, there are no significant differences. when examining the nucleotide distribution, significant differences are found for vertebrates, fungi, and plants, but not for arthropods. when using nucleotide pair frequencies, significant differences are found for all four groups. however, the results show no clear trends – there are no dinucleotide preferences that are the same in all four organism groups. the sets of proteins without signal peptides carrying a start codon intron were also subjected to further analysis, and here, some interesting differences were found. again, both the positive and the negative set consisted of proteins without signal peptides. the first thing we noticed was that proteins with start codon introns are on average shorter than other proteins, see table the differences are significant on the 5% level for vertebrates and fungi, and better than the % level for arthropods and plants. average length and amino acid frequencies for proteins without signal peptides, divided into proteins with and without start codon introns. where the difference for a particular amino acid is greater than %, the higher percentage is shown in boldface. the amino acid distributions were also calculated, and it was found that there are significant differences between proteins with and without start codon introns for all organism groups. the amino acid frequencies are shown in table the most conspicuous differences, seen in all four organism groups, are that lysine is more abundant in proteins with start codon introns than in other proteins, while serine is less abundant. in order to assess the conservation of start codon intron proteins, human genome proteins involved in a reciprocal best hit to mouse or fly were compared. while human proteins with a start codon intron on average have % identities in the global alignments with mouse, the other proteins have only %. for the alignments of human versus drosophila, the proteins with start codon introns had % identities, while the others had %. only rbhs where the human gene was predicted not to contain a signal peptide were included in these calculations. we also tested whether start codon intron positions are more conserved than other intron positions. all intron positions were mapped onto the global alignments, and in positions where the human protein had an intron it was tested if the mouse or fly protein also had an intron. in the comparison of human versus fly, there are rbhs with a human start codon intron. had a corresponding fly phase intron, and of these were also in a start codon position. for comparison, only of the human phase introns in other positions are conserved in fly. this difference is highly significant. in the comparison between human and mouse, where the overall conservation of phase intron positions is as high as %, no significant difference is seen. when looking at the functional annotations, a curious observation was made: in the sets of proteins with start codon introns, ribosomal proteins are overrepresented. this, of course, corresponds well with the fact that ribosomal proteins was the first place where start codon introns were observed. in the vertebrates, there are ribosomal proteins out of the start codon intron proteins, corresponding to 29%, while the background frequency in the non-start codon intron proteins without signal peptides is %. in the plants, there are out of against a background frequency of %, and in arthropods, there are out of against %. in fungi, there are only ribosomal proteins out of in the start codon intron set, but the overrepresentation is still significant. the descriptions of all start codon intron proteins from the genbank data sets can be seen in supplementary table s. in the genome sets, we found the same phenomenon: among human genes with start codon introns, there are % proteins marked "ribosomal" against a background frequency of %; in mouse, the frequencies are % against %, and in fly % against %. these differences are highly significant. since ribosomal proteins can be expected to be more conserved than other proteins, we investigated whether the higher conservation of proteins with start codon introns could be solely due to the high proportion of ribosomal proteins. the lists of rbhs were constrained to those where the human protein description did not contain the word "ribosomal," and the percent identities were compared again. the difference is still significant, although smaller. discussion when observing the start codon peak of introns in eukaryotic genes, the question naturally arose whether this was a biological phenomenon or some kind of artifact of gene finding. as mentioned in the description of the genbank data sets, we discarded genes where the evidence was marked as "not experimental." however, we did not limit the data set to genes marked "/evidence=experimental," as this yielded too few genes to carry out the statistical analysis. the majority of genes do not have an "/evidence" qualifier at all. the genbank data may therefore contain genes inferred by prediction. in the whole-genome data, on the other hand, we discarded the genes solely based on prediction and here, the start codon peak was still seen. this strongly suggests that the overabundance of start codon introns is a biological phenomenon. also, the observation that proteins carrying a start codon intron comprise a special class points in this direction. we have shown that proteins with start codon introns are relatively short, lysine-rich, serine-poor and evolutionarily conserved. in addition, we have shown that the start codon intron positions are often conserved between human and fly. during the peer review process it was suggested that our method for showing the excess of phase introns after the start codon could be questionable. the concern was that that the method of aligning the first aa of all protein sequences, while showing the start codon peak, might fail to detect some other peaks. the reason is that insertions and deletions during evolution would alter the distance between start codon and intron peak, thereby spreading the peak over several positions. in other words, peaks in other positions have been "diluted" over evolution while the start codon peak is retained simply because we use the start codon as "anchor point" for our alignment. this "dilution effect" would be enhanced by the fact that the n-terminal parts of proteins are often less conserved than other parts, with many indels. the reviewer suggested that we specifically analyzed proteins that have their n-terminals conserved over evolution to avoid this suggested effect. we did this by selecting from the list of rbhs between human and drosophila those global alignments where neither the human nor the fly sequence had gaps within the first positions. this yielded pairs, of which human sequences and fly sequences were predicted to be without signal peptides. we then repeated the intron position statistics for these sequences, and the result is very clear: the phase start codon peaks are even more conspicuous than those in figure, and there are no peaks in other positions. furthermore, the fact that we can still see the phase signal peptide peak in our start codon alignments despite the length variation of signal peptides suggests that the "dilution effect" does not completely wipe out peaks relatively close to the n-terminus, but only makes them broader. the reviewer suggested that the reason could be that proteins with signal peptides may have their n-terminals conserved over evolution, but this is not likely to be the case, since signal peptides are known to evolve rapidly. the function of the start codon intron may be to allow the 5' utr to participate in exon shuffling, so that different genes can exchange regulatory information. as described in the background section, phase introns are often found at the boundaries of evolutionary modules in proteins without signal peptides, and this effect is stronger in phylogenetically old proteins. this would correspond quite well with the 5' utr serving as an evolutionary module, separated from the protein coding domain by a phase intron at the start codon, especially in well-conserved proteins. if the start codon intron really is a boundary of an evolutionary module, one should expect that the second intron would often be a phase intron, too, since modules in exon shuffling most often have introns of the same phase in both ends. we tried to investigate this by counting the phases of the second introns in proteins where the first intron was a start codon intron, compared with proteins where the first intron was a phase intron in another position. in vertebrates and plants, there was indeed a tendency towards a higher proportion of second phase introns for the start codon intron set, but it was not significant. in arthropods there was no difference, and in fungi it was in fact lower. another explanation for the start codon introns could be that the nucleotide context of codons and provide a proto-splice site. the codon before is necessarily "atg," so the two nucleotides before the intron is "tg" instead of the canonical "ag." however, the "a" is not very well conserved, and "t" is the next most common nucleotide in the - position relative to the intron. also, in the evolutionarily conserved amino acids examined by sverdlov et al. in order to reconstruct ancestral proto-splice sites, methionine was slightly overrepresented immediately before a phase intron. consequently, the start codon could be part of a proto-splice site if the first position of codon is a "g". indeed, "g" is enriched in position + in eukaryotes. however, this enrichment is much higher in plants than in vertebrates or drosophila, so if this were to be the sole explanation for start codon introns, we should expect a much stronger start codon intron signal in plants than in animals, and this is not the case. on the other hand, the relatively weak start codon intron signal in plants could be explained within the proto-splice site model if there is a much higher frequency of proto-splice sites in the plant genes than in the vertebrate or arthropod genes as a whole. we have not checked whether this is the case. the 5' bias we observed in the position of introns within the non-vertebrate genes is in contrast to mourier and jeffares, who reported that introns in multicellular genomes are evenly distributed throughout genes. our fungal data set does contain unicellular organisms, but arthropods and plants do not. on the other hand, sakurai et al. and lin and zhang did find a 5' bias in virtually all genomes studied. interestingly, they even found 5' bias in human and mouse, where we did not observe it. the fact that ribosomal proteins are strongly overrepresented in start codon intron proteins is striking, because it is an example of an intron position being conserved in a functional class of proteins that are not related by descent. to be absolutely sure our homology reduction procedure had not let any pairs of homologous sequences through, we performed an all-versus-all global alignment of the sequences with start codon introns in all the four genbank organism groups. no pairs showed more than % identity in the global alignment. we also made phylogenetic trees of the start codon intron sets and found no clusters. plots of % identity versus alignment score and the trees can be seen in supplementary figure s section. another example of this is the position phase peak in drosophila. initially, we thought that it could be a gene finding artifact, since it was specifically seen in one genome. however, the extremely high proportion of cuticle constituents convinced us that this too is a biological phenomenon. it is also not entirely unknown in the literature: rondot et al., while describing two larval-pupal cuticle protein genes from the insect tenebrio molitor, found that both had a single intron within the signal peptide coding region. in addition to their own observation, they remarked that such an intron localization has been described in cuticle protein genes from many different species, including drosophila. again, note that this is not simply a gene family with a conserved intron position, since the peak is also seen in the heavily homology-reduced set. in order to test this further, we did an all-versus-all global alignment of the sequences in the arthropod peak and found no pairs with more than % identity.  the start codon peak of phase introns is so conspicuous that it is quite remarkable that it has only been described in a single sentence in the literature before. as mentioned above, yoshihama et al. saw the phenomenon in human ribosomal proteins, but to the best of our knowledge we are the first to provide a general investigation of it. in this paper, we have filled this gap in the description of intron positions in genes and argued that this phenomenon seems to be a biological reality and not simply an artifact of gene finding or alignment. 
in proteins with signal peptides, there is an overabundance of phase introns around the region of the signal peptide cleavage site. but in proteins without signal peptides, a novel phenomenon is observed: there is a sharp peak of phase intron positions immediately following the start codon, i.e. in addition, there is a peak of phase introns at position in drosophila genes with signal peptides, predominantly representing cuticle proteins. proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. there is an overabundance of phase introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. we give a detailed description of these start codon introns and the proteins that contain them. a knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. despite this, there has been relatively little focus on the distribution of intron positions in genes.
 a knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. despite this, there has been relatively little focus on the distribution of intron positions in genes.  in proteins with signal peptides, there is an overabundance of phase introns around the region of the signal peptide cleavage site. this has been described before. but in proteins without signal peptides, a novel phenomenon is observed: there is a sharp peak of phase intron positions immediately following the start codon, i.e. between codons and this effect is seen in a wide range of eukaryotes: vertebrates, arthropods, fungi, and flowering plants. proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. in addition, there is a peak of phase introns at position in drosophila genes with signal peptides, predominantly representing cuticle proteins.  there is an overabundance of phase introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. we give a detailed description of these start codon introns and the proteins that contain them. 