BACKGROUND
overlapping translated open reading frames  are usually associated with genomes under selection pressure to remain compact, such as those of viruses.
however, such overlaps also exist in mammals.
for example in human, an exon is shared by the ink4a and arf genes and is translated in different frames over  <dig> bases  <cit> .
similarly, a transcript fusion between the human eif4ebp <dig> and mask genes results in the translation of  <dig> bases in two different frames  <cit> .
an alternative splice variant of insulin-like growth factor  <dig> , called mechano-growth-factor , contains a frameshift which leads to translation of overlapping reading frames  <cit> .
expression profiling of igf- <dig> and mgf indicates that the variants have distinct physiological roles.
the best-characterised case of overlapping torfs in mammals is that of xl-alpha-s. this is a splice variant of a g protein alpha subunit, derived from the gnas complex locus, which is expressed in neuroendocrine tissues and other tissues.
the first exon of xl-alpha-s contains a downstream orf which is frameshifted + <dig> relative to the xl-alpha-s initiator codon.
this orf gives rise to an entirely different protein called alex, which is  <dig> amino acids long in rat  <cit> .
remarkably, xl-alpha-s and alex interact, and this interaction can be disrupted by an insertion polymorphism in humans.
the polymorphism leads to enhanced receptor-mediated camp formation in platelets and fibroblasts, increased trauma-related bleeding tendency, and in two families neurological problems and brachydactyly were observed  <cit> .
furthermore, the xl-alpha-s and alex orfs may extend in the 5' direction for several hundred nucleotides more  <cit> , raising the possibility that longer variants of xl-alpha-s and alex exist.
although the alex termination codon lies well within  <dig> base pairs  of the next 3' splice junction, the xl-alpha-s transcript does not appear to be degraded according to the usual rules for nonsense-mediated decay  <cit> .
figure  <dig> summarises these cases of overlapping mammalian torfs.
during our own in silico comparative studies of entire translated human, mouse and rat genomes, we frequently observed overlapping torfs conserved at the amino acid level.
in an effort to explore this relatively uncharacterised aspect of gene structure and evolution, we screened for additional alex-like cases in human and rodents using a bioinformatics approach.
specifically, we searched human, mouse and rat transcripts for frameshifted conserved torfs.
conservation of such sequences at the amino acid level may reflect a functional role.
a related comparative genomics approach, supported by simulation-based statistics, has recently been published  <cit> .
based on conservation between human and mouse, chung et al.
convincingly demonstrate that these frameshifted orfs  are highly unlikely to occur by chance.
in our study, the term 'matreshka' was coined to describe the overlapping torfs, in analogy with russian dolls, as one protein can be thought of as 'hiding' another.
it should be kept in mind, however, that all matreshkas reported here are observations based on mrna sequence.
translation of these sequences in vivo has not yet been experimentally confirmed.
RESULTS
matreshkas are defined here as overlapping, frameshifted orfs  in transcripts, which are well conserved at the amino acid level.
matreshkas may potentially represent alternative translation products like alex, or suggest the existence of functional frameshifted splice variants.
to obtain matreshka predictions, we translated in silico all frame + <dig> and - <dig> orfs from known human, mouse and rat "parent" transcripts, and applied a conservation filter .
open reading frame translation
the first step was to build human-mouse-rat ortholog triplets, from which frame + <dig> and - <dig> orfs were extracted .
ortholog sets were based on a variation of best reciprocal blastp hits, which favoured hits with a higher percentage identity .
this method yielded  <dig> triplets.
the coding nucleotide sequence was retrieved for each ortholog, and all translated orfs  at least  <dig> amino acids  long were extracted computationally.
the matreshka identification pipeline was run twice, each time varying the torf extraction method.
in an effort to identify alex-like cases, the first run retained only torfs beginning with methionine .
the second run was somewhat more comprehensive, including translated orfs starting with any amino acid .
it should be kept in mind that although the torfm set is contained within the torfx set, the derived matreshka sets only partially overlap, due to the properties of the conservation filter.
the term 'torf' is used here to collectively refer to torfm and torfx.
this early stage in the analysis, after torf extraction but before the conservation filter, already yielded surprising results: torfs beginning with methionine  were found to be ~10-fold rarer in frame - <dig> than in frame + <dig> .
this effect was also seen for torfx, but in a much less pronounced way .
the pattern was mirrored by human codon usage frequencies : stop codons are more frequent in frame - <dig> than frame + <dig>  resulting in fewer frame - <dig> torfs.
in addition, atg codons are ~5-fold rarer in frame - <dig> compared to + <dig>  accounting further for the observed scarcity of frame - <dig> torfm.
in the arf study  <cit> , the difference between the two frames was echoed in simulated alignments.
the importance of this apparent suppression of torfm may be part of a mechanism to minimize the impact of translation initiation errors.
why there should be a difference between frames + <dig> and - <dig>  however, is unclear.
perhaps an alternative mechanism already exists to prevent erroneous frame + <dig> translation initiation.
examination of amino acid sequences in the parent frame sheds some light on how stop codons can occur in other frames.
if, for instance, a tyrosine  is followed by an aspartic acid , the stop codon tga occurs in frame - <dig> if, and only if, the codon used for tyrosine is tat.
overall, codon tat is used for tyrosine in 45% of the cases in human genes.
however, when tyrosine is directly followed by aspartic acid in a coding sequence, tat occurs in 75% of the cases to code for tyrosine .
thus, the codon which leads to a stop codon in frame - <dig> is favoured.
in order to explore whether this observation can be generalized, we calculated a  <dig> by  <dig> bi-codon usage matrix for a set of human coding sequences .
table  <dig> shows as an example the combinations of two consecutive amino acids , where the second amino acid is aspartic acid .
interestingly, there is a trend for codons to be favored which lead to a stop codon in the frame - <dig>  in some cases leading to highly significant probability values .
the same effect can be observed when the second amino acid in the bi-codon is glutamic acid .
in contrast, if the second amino acid is lysine or asparagine, the opposite trend appears .
this may be due to interacting effects on the complementary nucleotide strand, and illustrates the complexity of bi-codon effects on the appearance of stop codons in frame - <dig>  note that our analysis did not take into account the base following the stop codon, which can have a significant effect on efficiency of termination of translation  <cit> .
conservation filter
having extracted all torfs, the conservation filter was then applied.
within each ortholog triplet, all human frame + <dig> and - <dig> torfs were compared in pairwise global alignments to all those computed from mouse and from rat.
well-conserved torf triplets which passed the length similarity and identity criteria were retained for further analysis as candidate matreshkas.
matreshkas derived from torfm are denoted here as matreshkam, those derived from torfx are denoted matreshkax, while a lack of subscript refers to both matreshka sets collectively.
the breakdown of torf and matreshka statistics by frame is given in table  <dig>
of the  <dig> matreshkax ,  <dig> are at least partially redundant with the matreshkam , of which there are  <dig> in total.
the matreshkam total  is smaller than the number of redundant matreshkax , because the longer matreshkax can contain several of the shorter matreshkam.
frame - <dig> torfs were filtered out more effectively than frame + <dig> : the proportion of frame - <dig> torfx dropped from 45% to 25% of the total after conservation filtering, and from 9% to 6% for torfm.
the greater stringency of the conservation filter on frame - <dig> torfs was expected, and is a consequence of the variation in the third base of each parent codon: this can be demonstrated in a simple exercise by combining all possible nucleotide triplet pairs.
for a given amino acid in frame  <dig>  on average ~ <dig> different amino acids can potentially be coded for in frame + <dig>  while ~ <dig> can be coded for in frame - <dig> .
the scarcity of frame - <dig> torfm, combined with the greater efficacy of the conservation filter, resulted in very few  matreshkam in frame - <dig>  for reference, alex lies in frame + <dig>
no matreshkas were found corresponding to alex/xl-alpha-s  <cit> , ink4a/arf  <cit>  or 4e-bp3/mask  <cit>  because they were not represented in the ortholog triplets  and in the case of igf <dig>  <cit>  because the region which is translated in two frames is short .
as recovery of alex-like entities but not alex itself was our primary goal, we used stringent conservation filters that alex could not have passed: human alex is only 53% and 55% identical at the amino acid level to mouse and rat alex, respectively  <cit> , while our conservation threshold was set at 60%.
this relatively high threshold was necessary partly because the exploratory nature of the project made clear-cut examples necessary, but was also based on separate benchmark studies we made on the chemokine family.
chemokines are typically quite poorly conserved, and as such provide an indication of how much sequences may diverge while retaining similar functions.
for instance, half of mouse-human chemokine orthologs in homologene  <cit>  have an amino acid sequence identity of 60–70% .
since a 60% cut-off seems suitable for this family, and we were interested in detecting possible new peptide ligands, it was chosen as a threshold for the matreshkas.
a number of matreshkas mapped to alternative, frameshifted splice variants .
in some cases, there is evidence that these variants are functionally distinct.
among these are the paired box gene  <dig>  isoform c  <cit> , and x-box protein  isoform u  <cit> .
given the nucleotide sequence similarity between transcripts in orthologous triplets, conserved translation products in alternative reading frames would be expected to occur by chance.
additional parameters were required to differentiate likely matreshkas from false positives.
generally, longer matreshkas are less likely to arise by chance, therefore matreshka length was chosen as a first parameter for selection.
a second useful parameter gave an approximate measure of the selection pressure maintaining a potential matreshka: this was the number of amino acid positions where a stop codon could have arisen, truncating the matreshka but leaving the parent  protein sequence unaffected , denoted here nstop.
furthermore, we estimated a probability of seeing nstop given the parent amino acid sequence, and considering codon bias , but not taking into account the effects of bi-codon bias mentioned above .
to gain clues as to the possible function of matreshkas, all matreshka protein sequences were analysed with functional motif and signal peptide prediction programs.
matreshka nucleotide sequences were also screened for tandem repeats, to exclude artifactual sequences which pass the length filter .
matreshkas with tandem repeats in any one of the three organisms were discarded from in-depth analysis, even though alex itself was found to contain such repeats.
in addition, matreshkam nucleotide sequences were scanned for consensus kozak translation initiation motifs  <cit> , to help determine whether leaky ribosomal scanning  <cit>  may be occuring.
to estimate how frequently kozak motifs appear by chance, we scanned the  <dig> human coding sequences used for building matreshkas, and counted how frequently atg occurred versus full kozak sequences .
on average, ~ <dig> atgs, of which ~ <dig>  were kozak motifs, were observed per transcript.
of the  <dig> matreshkam, only  <dig>  have a kozak motif exactly at the start codon .
however, of the  <dig> longest matreshkam,  <dig> have a kozak motif exactly at the start codon, which is a significant enrichment of true positives .
the matreshka nstop versus length distribution can be seen in fig.
<dig>  with outliers of interest highlighted .
details on the highlighted matreshkas can be found in table  <dig>  some matreshkas can be extended into 5' or 3' untranslated regions  or  into flanking genomic sequence .
of particular interest is the thpo matreshka, which is of the rare methionine-start, frame - <dig> variety.
the matreshkas in table  <dig> which don't begin with methionine are unlikely to represent complete proteins, since the human matreshka sequence either contains no methionines  or methionines only very close to the 3' end of the matreshka .
as with alex and virtually all matreshka predictions, none of the candidates in table  <dig> contain known protein functional motifs, as ascertained by pattern matching to prosite motifs.
the following sections describe matreshkas of particular interest.
case study 1: matreshka framing super conserved receptor expressed in brain  <dig>
the first example presented here is of a particularly large matreshka associated with the gpr <dig> transcript, also called super conserved receptor expressed in brain  <dig>  gpr <dig> is an aminergic g-protein coupled receptor, cloned from human brain cdna and part of a family of three members which are highly conserved between vertebrates  <cit> .
this single-exon coding sequence is entirely contained within a frame - <dig> matreshkax, which begins and ends in the flanking utrs .
the matreshkax varies in length between  <dig> and  <dig> amino acids depending on the species, and in the refseq coding region alone has between  <dig>  and  <dig>  positions where a stop codon could have truncated the matreshkax, suggesting selection pressure to maintain it.
the probability in human of a stop codon truncating the matreshka, pstop, was estimated at  <dig> .
it seems possible from our analysis that the high level of conservation of this gene may be necessary to maintain the protein sequences of both gpr <dig> and the matreshka.
figure 5b shows the matreshkax amino acid sequence, which like alex is basic  and enriched in proline residues .
the gpr <dig> matreshka is unlikely to be translated from a leaky scanning event  <cit> , since it contains only one methionine residue  <dig> amino acids from its c-terminus.
it may be part of the exon of a longer, unknown transcript, in which case the ag straddling its 5' end could be a canonical splice signal.
case study 2: matreshka contained in thrombopietin
thrombopoietin is the major regulator of platelet production by megakaryocytes, with associated disorders including thrombocytosis and thrombocytopenia.
splice variant  <dig> of thpo contains a matreshka of the scarce frame - <dig> methionine-start type, which in human is  <dig> amino acids long, and covers 72% of the length of thpo .
it contains  <dig> positions where a stop codon could have occurred without changing the parent refseq protein sequence .
for comparison, in frame + <dig> there are  <dig> positions where a stop codon could occur, and  <dig> stop codons actually did.
like alex, the thpo matreshka is proline-rich .
the genomic region upstream of the matreshkam start was computationally scanned for transcription start sites , in case it is in fact derived from a separate transcript overlapping with the thpo sequence: none was found.
the matreshkam start is  <dig> nucleotides downstream from the parent start, and the initiator atg lies in a strong kozak context  in all three organisms.
this suggests that the matreshka could be produced from a leaky ribosomal scanning event  <cit> .
an important indication, however, that the thpo matreshkam may be translated not from a leaky scanning event, but instead as part of a frameshifted splice variant, came from a blast search of the matreshkam against the uniprot protein database  <cit> .
a 100% identity match was found against the translation of the 'nirs' thpo splice variant.
no function specific to the nirs thpo appears to be documented.
this variant contains a frameshift downstream of the matreshkam start, which effectively places the 3' half of the transcript in the same frame as the matreshkam .
a snp  <cit>  exists which can generate a stop codon in  the matreshkam at the 45th amino acid .
the snp replaces a glycine with a glutamate at position  <dig> in thpo itself, on a loop between an alpha-helix and a beta sheet.
gly <dig> is within the conserved, 184aa long erythropoietin-thrombopoietin domain in the n-terminal portion of thpo.
this domain is responsible for binding to thpo's receptor, mpl .
three independent site-directed mutagenesis studies have been carried out to determine which residues are necessary for binding to mpl, based on 3d structural data  <cit> .
a total of  <dig> amino acids were determined to be important for binding, none of which lie within 15aa of gly <dig>  it therefore seems unlikely that the snp affects thpo function.
also of note are two splice variants , which retain the snp and shorten the matreshkam by  <dig> and  <dig> aa, respectively.
case study iii: matreshka contained in platelet glycoprotein iiia
platelet glycoprotein iiia  is part of the gpiib/gpiiia complex, which mediates platelet aggregation by acting as a receptor for fibrinogen.
a frame + <dig> matreshkam  spans  <dig> exons in the itgb <dig> transcript.
it is  <dig> amino acids long in human, and has strong kozak sequences and a signal peptide prediction in all three organisms , coupled with dibasic cleavage sites clustered at the n and c-termini which could potentially produce conserved 'active' secreted peptides .
such characteristics are typical of gpcr ligands, for example  <cit> , which are of great value for drug discovery.
from an evolutionary perspective, it seems plausible that such a secreted peptide could 'signal' the expression of itgb <dig>  since any putative receptor would be able to freely co-evolve with the matreshka, according to the sequence requirements of itgb <dig> itself.
the significance of the matreshka's kozak motif is underlined by the absence of a kozak motif for the parent sequence: the first motif in the itgb <dig> frame occurs  <dig> nucleotides downstream of the annotated start codon.
this suggests that leaky ribosomal scanning is occurring in the parent frame, and may increase the likelihood that the matreshkam  is also translated from leaky scanning.
these are by no means the only interesting predictions.
the largest matreshka discovered is 448aa long in human, although it has the potential to reach up to 500aa in mouse .
it is derived from the kcnk <dig> potassium channel, a member of the 2-pore domain superfamily of background k+ channels.
the g protein-coupled receptor  <dig>  the gamma subunit of a dna directed polymerase, taf <dig> rna polymerase ii, and forkhead box a <dig> also contain substantial size matreshkas .
these matreshkas may represent alternative proteins, in the way that alex is, or be translated as part of a frameshifted splice variant .
conservation measurements have previously been used to support the functional importance of frameshifted splice variants  <cit> .
our findings suggest that other conserved frameshifted splice variants, such as caspase  <dig> isoform beta, are biologically relevant .
in a related study by chung et al  <cit> , alternative reading frames  were searched for using a comparative genomics approach in human and mouse.
a conservative list of  <dig> arf-containing genes was derived from elegant simulation-based statistical and modelling methods.
of these  <dig> genes,  <dig> genes were included in our matreshka set , although the arf and matreshka sequences were not compared directly, as the former have not been published.
our study had a broader scope, including non-methionine-start reading frames which form the bulk of the longest matreshkas .
given the stringent length threshold chosen by chung et al., our analysis probably has a much greater representation of - <dig> frame matreshkas .
all matreshka sequences have been provided as suppplementary data , and can be filtered according to our calculated p values .
CONCLUSIONS
the genome-wide study reported here provides evidence for the existence of potentially dual coding sense-frames in a number of mammalian transcripts.
future studies should aim to determine actual proof-of-translation by raising antibodies against matreshkas, and probing cells or tissues where the mrnas are known to be expressed.
alternatively genetic mouse models could be generated which would knock-out the putative matreshka while leaving the parent refseq sequence intact, thus enabling phenotypic analysis.
future studies could also explore the potential for overlaps between torfs on the antisense strands.
indeed, genomic mapping of full-length mouse cdnas has revealed transcriptional forests in which overlap of coding sequences on the sense and antisense strands occurs  <cit> .

 overlapping translated open reading frames are usually associated with genomes under selection pressure to remain compact, such as those of viruses. however, such overlaps also exist in mammals. for example in human, an exon is shared by the ink4a and arf genes and is translated in different frames over bases. similarly, a transcript fusion between the human eif4ebp and mask genes results in the translation of bases in two different frames. an alternative splice variant of insulin-like growth factor, called mechano-growth-factor, contains a frameshift which leads to translation of overlapping reading frames. expression profiling of igf- and mgf indicates that the variants have distinct physiological roles. the best-characterised case of overlapping torfs in mammals is that of xl-alpha-s. this is a splice variant of a g protein alpha subunit, derived from the gnas complex locus, which is expressed in neuroendocrine tissues and other tissues. the first exon of xl-alpha-s contains a downstream orf which is frameshifted + relative to the xl-alpha-s initiator codon. this orf gives rise to an entirely different protein called alex, which is amino acids long in rat. remarkably, xl-alpha-s and alex interact, and this interaction can be disrupted by an insertion polymorphism in humans. the polymorphism leads to enhanced receptor-mediated camp formation in platelets and fibroblasts, increased trauma-related bleeding tendency, and in two families neurological problems and brachydactyly were observed. furthermore, the xl-alpha-s and alex orfs may extend in the 5' direction for several hundred nucleotides more, raising the possibility that longer variants of xl-alpha-s and alex exist. although the alex termination codon lies well within base pairs of the next 3' splice junction, the xl-alpha-s transcript does not appear to be degraded according to the usual rules for nonsense-mediated decay. figure summarises these cases of overlapping mammalian torfs. during our own in silico comparative studies of entire translated human, mouse and rat genomes, we frequently observed overlapping torfs conserved at the amino acid level. in an effort to explore this relatively uncharacterised aspect of gene structure and evolution, we screened for additional alex-like cases in human and rodents using a bioinformatics approach. specifically, we searched human, mouse and rat transcripts for frameshifted conserved torfs. conservation of such sequences at the amino acid level may reflect a functional role. a related comparative genomics approach, supported by simulation-based statistics, has recently been published. based on conservation between human and mouse, chung et al. convincingly demonstrate that these frameshifted orfs are highly unlikely to occur by chance. in our study, the term'matreshka' was coined to describe the overlapping torfs, in analogy with russian dolls, as one protein can be thought of as'hiding' another. it should be kept in mind, however, that all matreshkas reported here are observations based on mrna sequence. translation of these sequences in vivo has not yet been experimentally confirmed.  matreshkas are defined here as overlapping, frameshifted orfs in transcripts, which are well conserved at the amino acid level. matreshkas may potentially represent alternative translation products like alex, or suggest the existence of functional frameshifted splice variants. to obtain matreshka predictions, we translated in silico all frame + and - orfs from known human, mouse and rat "parent" transcripts, and applied a conservation filter. open reading frame translation the first step was to build human-mouse-rat ortholog triplets, from which frame + and - orfs were extracted. ortholog sets were based on a variation of best reciprocal blastp hits, which favoured hits with a higher percentage identity. this method yielded triplets. the coding nucleotide sequence was retrieved for each ortholog, and all translated orfs at least amino acids long were extracted computationally. the matreshka identification pipeline was run twice, each time varying the torf extraction method. in an effort to identify alex-like cases, the first run retained only torfs beginning with methionine. the second run was somewhat more comprehensive, including translated orfs starting with any amino acid. it should be kept in mind that although the torfm set is contained within the torfx set, the derived matreshka sets only partially overlap, due to the properties of the conservation filter. the term'torf' is used here to collectively refer to torfm and torfx. this early stage in the analysis, after torf extraction but before the conservation filter, already yielded surprising results: torfs beginning with methionine were found to be ~10-fold rarer in frame - than in frame +. this effect was also seen for torfx, but in a much less pronounced way. the pattern was mirrored by human codon usage frequencies: stop codons are more frequent in frame - than frame + resulting in fewer frame - torfs. in addition, atg codons are ~5-fold rarer in frame - compared to + accounting further for the observed scarcity of frame - torfm. in the arf study, the difference between the two frames was echoed in simulated alignments. the importance of this apparent suppression of torfm may be part of a mechanism to minimize the impact of translation initiation errors. why there should be a difference between frames + and - however, is unclear. perhaps an alternative mechanism already exists to prevent erroneous frame + translation initiation. examination of amino acid sequences in the parent frame sheds some light on how stop codons can occur in other frames. if, for instance, a tyrosine is followed by an aspartic acid, the stop codon tga occurs in frame - if, and only if, the codon used for tyrosine is tat. overall, codon tat is used for tyrosine in 45% of the cases in human genes. however, when tyrosine is directly followed by aspartic acid in a coding sequence, tat occurs in 75% of the cases to code for tyrosine. thus, the codon which leads to a stop codon in frame - is favoured. in order to explore whether this observation can be generalized, we calculated a by bi-codon usage matrix for a set of human coding sequences. table shows as an example the combinations of two consecutive amino acids, where the second amino acid is aspartic acid. interestingly, there is a trend for codons to be favored which lead to a stop codon in the frame - in some cases leading to highly significant probability values. the same effect can be observed when the second amino acid in the bi-codon is glutamic acid. in contrast, if the second amino acid is lysine or asparagine, the opposite trend appears. this may be due to interacting effects on the complementary nucleotide strand, and illustrates the complexity of bi-codon effects on the appearance of stop codons in frame - note that our analysis did not take into account the base following the stop codon, which can have a significant effect on efficiency of termination of translation. conservation filter having extracted all torfs, the conservation filter was then applied. within each ortholog triplet, all human frame + and - torfs were compared in pairwise global alignments to all those computed from mouse and from rat. well-conserved torf triplets which passed the length similarity and identity criteria were retained for further analysis as candidate matreshkas. matreshkas derived from torfm are denoted here as matreshkam, those derived from torfx are denoted matreshkax, while a lack of subscript refers to both matreshka sets collectively. the breakdown of torf and matreshka statistics by frame is given in table of the matreshkax, are at least partially redundant with the matreshkam, of which there are in total. the matreshkam total is smaller than the number of redundant matreshkax, because the longer matreshkax can contain several of the shorter matreshkam. frame - torfs were filtered out more effectively than frame +: the proportion of frame - torfx dropped from 45% to 25% of the total after conservation filtering, and from 9% to 6% for torfm. the greater stringency of the conservation filter on frame - torfs was expected, and is a consequence of the variation in the third base of each parent codon: this can be demonstrated in a simple exercise by combining all possible nucleotide triplet pairs. for a given amino acid in frame on average ~ different amino acids can potentially be coded for in frame + while ~ can be coded for in frame -. the scarcity of frame - torfm, combined with the greater efficacy of the conservation filter, resulted in very few matreshkam in frame - for reference, alex lies in frame + no matreshkas were found corresponding to alex/xl-alpha-s, ink4a/arf or 4e-bp3/mask because they were not represented in the ortholog triplets and in the case of igf because the region which is translated in two frames is short. as recovery of alex-like entities but not alex itself was our primary goal, we used stringent conservation filters that alex could not have passed: human alex is only 53% and 55% identical at the amino acid level to mouse and rat alex, respectively, while our conservation threshold was set at 60%. this relatively high threshold was necessary partly because the exploratory nature of the project made clear-cut examples necessary, but was also based on separate benchmark studies we made on the chemokine family. chemokines are typically quite poorly conserved, and as such provide an indication of how much sequences may diverge while retaining similar functions. for instance, half of mouse-human chemokine orthologs in homologene have an amino acid sequence identity of 60–70%. since a 60% cut-off seems suitable for this family, and we were interested in detecting possible new peptide ligands, it was chosen as a threshold for the matreshkas. a number of matreshkas mapped to alternative, frameshifted splice variants. in some cases, there is evidence that these variants are functionally distinct. among these are the paired box gene isoform c, and x-box protein isoform u. given the nucleotide sequence similarity between transcripts in orthologous triplets, conserved translation products in alternative reading frames would be expected to occur by chance. additional parameters were required to differentiate likely matreshkas from false positives. generally, longer matreshkas are less likely to arise by chance, therefore matreshka length was chosen as a first parameter for selection. a second useful parameter gave an approximate measure of the selection pressure maintaining a potential matreshka: this was the number of amino acid positions where a stop codon could have arisen, truncating the matreshka but leaving the parent protein sequence unaffected, denoted here nstop. furthermore, we estimated a probability of seeing nstop given the parent amino acid sequence, and considering codon bias, but not taking into account the effects of bi-codon bias mentioned above. to gain clues as to the possible function of matreshkas, all matreshka protein sequences were analysed with functional motif and signal peptide prediction programs. matreshka nucleotide sequences were also screened for tandem repeats, to exclude artifactual sequences which pass the length filter. matreshkas with tandem repeats in any one of the three organisms were discarded from in-depth analysis, even though alex itself was found to contain such repeats. in addition, matreshkam nucleotide sequences were scanned for consensus kozak translation initiation motifs, to help determine whether leaky ribosomal scanning may be occuring. to estimate how frequently kozak motifs appear by chance, we scanned the human coding sequences used for building matreshkas, and counted how frequently atg occurred versus full kozak sequences. on average, ~ atgs, of which ~ were kozak motifs, were observed per transcript. of the matreshkam, only have a kozak motif exactly at the start codon. however, of the longest matreshkam, have a kozak motif exactly at the start codon, which is a significant enrichment of true positives. the matreshka nstop versus length distribution can be seen in fig. with outliers of interest highlighted. details on the highlighted matreshkas can be found in table some matreshkas can be extended into 5' or 3' untranslated regions or into flanking genomic sequence. of particular interest is the thpo matreshka, which is of the rare methionine-start, frame - variety. the matreshkas in table which don't begin with methionine are unlikely to represent complete proteins, since the human matreshka sequence either contains no methionines or methionines only very close to the 3' end of the matreshka. as with alex and virtually all matreshka predictions, none of the candidates in table contain known protein functional motifs, as ascertained by pattern matching to prosite motifs. the following sections describe matreshkas of particular interest. case study 1: matreshka framing super conserved receptor expressed in brain the first example presented here is of a particularly large matreshka associated with the gpr transcript, also called super conserved receptor expressed in brain gpr is an aminergic g-protein coupled receptor, cloned from human brain cdna and part of a family of three members which are highly conserved between vertebrates. this single-exon coding sequence is entirely contained within a frame - matreshkax, which begins and ends in the flanking utrs. the matreshkax varies in length between and amino acids depending on the species, and in the refseq coding region alone has between and positions where a stop codon could have truncated the matreshkax, suggesting selection pressure to maintain it. the probability in human of a stop codon truncating the matreshka, pstop, was estimated at. it seems possible from our analysis that the high level of conservation of this gene may be necessary to maintain the protein sequences of both gpr and the matreshka. figure 5b shows the matreshkax amino acid sequence, which like alex is basic and enriched in proline residues. the gpr matreshka is unlikely to be translated from a leaky scanning event, since it contains only one methionine residue amino acids from its c-terminus. it may be part of the exon of a longer, unknown transcript, in which case the ag straddling its 5' end could be a canonical splice signal. case study 2: matreshka contained in thrombopietin thrombopoietin is the major regulator of platelet production by megakaryocytes, with associated disorders including thrombocytosis and thrombocytopenia. splice variant of thpo contains a matreshka of the scarce frame - methionine-start type, which in human is amino acids long, and covers 72% of the length of thpo. it contains positions where a stop codon could have occurred without changing the parent refseq protein sequence. for comparison, in frame + there are positions where a stop codon could occur, and stop codons actually did. like alex, the thpo matreshka is proline-rich. the genomic region upstream of the matreshkam start was computationally scanned for transcription start sites, in case it is in fact derived from a separate transcript overlapping with the thpo sequence: none was found. the matreshkam start is nucleotides downstream from the parent start, and the initiator atg lies in a strong kozak context in all three organisms. this suggests that the matreshka could be produced from a leaky ribosomal scanning event. an important indication, however, that the thpo matreshkam may be translated not from a leaky scanning event, but instead as part of a frameshifted splice variant, came from a blast search of the matreshkam against the uniprot protein database. a 100% identity match was found against the translation of the'nirs' thpo splice variant. no function specific to the nirs thpo appears to be documented. this variant contains a frameshift downstream of the matreshkam start, which effectively places the 3' half of the transcript in the same frame as the matreshkam. a snp exists which can generate a stop codon in the matreshkam at the 45th amino acid. the snp replaces a glycine with a glutamate at position in thpo itself, on a loop between an alpha-helix and a beta sheet. gly is within the conserved, 184aa long erythropoietin-thrombopoietin domain in the n-terminal portion of thpo. this domain is responsible for binding to thpo's receptor, mpl. three independent site-directed mutagenesis studies have been carried out to determine which residues are necessary for binding to mpl, based on 3d structural data. a total of amino acids were determined to be important for binding, none of which lie within 15aa of gly it therefore seems unlikely that the snp affects thpo function. also of note are two splice variants, which retain the snp and shorten the matreshkam by and aa, respectively. case study iii: matreshka contained in platelet glycoprotein iiia platelet glycoprotein iiia is part of the gpiib/gpiiia complex, which mediates platelet aggregation by acting as a receptor for fibrinogen. a frame + matreshkam spans exons in the itgb transcript. it is amino acids long in human, and has strong kozak sequences and a signal peptide prediction in all three organisms, coupled with dibasic cleavage sites clustered at the n and c-termini which could potentially produce conserved'active' secreted peptides. such characteristics are typical of gpcr ligands, for example, which are of great value for drug discovery. from an evolutionary perspective, it seems plausible that such a secreted peptide could'signal' the expression of itgb since any putative receptor would be able to freely co-evolve with the matreshka, according to the sequence requirements of itgb itself. the significance of the matreshka's kozak motif is underlined by the absence of a kozak motif for the parent sequence: the first motif in the itgb frame occurs nucleotides downstream of the annotated start codon. this suggests that leaky ribosomal scanning is occurring in the parent frame, and may increase the likelihood that the matreshkam is also translated from leaky scanning. these are by no means the only interesting predictions. the largest matreshka discovered is 448aa long in human, although it has the potential to reach up to 500aa in mouse. it is derived from the kcnk potassium channel, a member of the 2-pore domain superfamily of background k+ channels. the g protein-coupled receptor the gamma subunit of a dna directed polymerase, taf rna polymerase ii, and forkhead box a also contain substantial size matreshkas. these matreshkas may represent alternative proteins, in the way that alex is, or be translated as part of a frameshifted splice variant. conservation measurements have previously been used to support the functional importance of frameshifted splice variants. our findings suggest that other conserved frameshifted splice variants, such as caspase isoform beta, are biologically relevant. in a related study by chung et al, alternative reading frames were searched for using a comparative genomics approach in human and mouse. a conservative list of arf-containing genes was derived from elegant simulation-based statistical and modelling methods. of these genes, genes were included in our matreshka set, although the arf and matreshka sequences were not compared directly, as the former have not been published. our study had a broader scope, including non-methionine-start reading frames which form the bulk of the longest matreshkas. given the stringent length threshold chosen by chung et al., our analysis probably has a much greater representation of - frame matreshkas. all matreshka sequences have been provided as suppplementary data, and can be filtered according to our calculated p values.  the genome-wide study reported here provides evidence for the existence of potentially dual coding sense-frames in a number of mammalian transcripts. future studies should aim to determine actual proof-of-translation by raising antibodies against matreshkas, and probing cells or tissues where the mrnas are known to be expressed. alternatively genetic mouse models could be generated which would knock-out the putative matreshka while leaving the parent refseq sequence intact, thus enabling phenotypic analysis. future studies could also explore the potential for overlaps between torfs on the antisense strands. indeed, genomic mapping of full-length mouse cdnas has revealed transcriptional forests in which overlap of coding sequences on the sense and antisense strands occurs. 
exceptionally, a single nucleotide sequence can be translated in vivo in two different frames to yield distinct proteins. our method searched human, mouse and rat transcripts in frames + and - for orfs which are unusually well conserved at the amino acid level.
 exceptionally, a single nucleotide sequence can be translated in vivo in two different frames to yield distinct proteins. in the case of the g-protein alpha subunit xl-alpha-s transcript, a frameshifted open reading frame in exon is translated to yield a structurally distinct protein called alex, which plays a role in platelet aggregation and neurological processes. we carried out a novel bioinformatics screen for other possible dual-frame translated sequences, based on comparative genomics.  our method searched human, mouse and rat transcripts in frames + and - for orfs which are unusually well conserved at the amino acid level. we name these conserved frameshifted overlapping orfs'matreshkas' to reflect their nested character. select findings of our analysis revealed that the g-protein coupled receptor gpr is entirely contained within a frame - matreshka, thrombopoietin contains a matreshka which spans ~70% of its length, platelet glycoprotein iiia contains a matreshka with the predicted characteristics of a secreted peptide hormone, while the potassium channel kcnk contains a matreshka spanning > amino acids.  although the in vivo existence of translated matreshkas has not been experimentally verified, this genome-wide analysis provides strong evidence that substantial overlapping coding sequences exist in a number of human and rodent transcripts. 