BACKGROUND
polycomb group  proteins are epigenetic gene regulators implicated in important cellular and developmental processes.
in animals, pcg proteins were primarily known for their role in maintaining cell identity during the establishment of the body plan  <cit> , but recently they have also been implicated in other important processes like stem cell self-renewal and cancer  <cit> .
in plants, the pcg proteins are involved in the repression of flowering during vegetative development  <cit> , seed development  <cit> , and in the vernalization response  <cit> .
hence, pcg proteins play an essential role in the proper development of multicellular organisms.
biochemical and genetic characterizations of pcg proteins have revealed that they exist in distinct multi-protein complexes  <cit> , of which the two best characterized are the pcg repressive complex  <dig> and prc <dig>  the core components of prc <dig> are conserved throughout higher eukaryotes.
the complex mediates histone h <dig> methylation at lysine  <dig>  <cit> .
similar complexes have been found in caenorhabditis elegans  <cit> , vertebrates  <cit>  and plants  <cit> .
unlike prc <dig>  prc1-like complexes have only been described in drosophila and vertebrates.
drosophila prc <dig> core complex contains posterior sex combs , polyhomeotic , drosophila ring finger protein  <dig> or sex combs extra  and polycomb   <cit> .
the complex can inhibit in vitro both chromatin remodeling by the human switch/sucrose nonfermentable  complex  <cit>  and transcription of a chromatin template by rna polymerase ii  <cit> .
the human prc <dig> complex is composed of b lymphoma mo-mlv insertion region  <dig> homolog , ph <dig>  pc <dig>  and the ring finger proteins , homologs of drosophila psc, ph, pc, and dring, respectively.
human bmi <dig>  ring1a and ring1b proteins form an e <dig> ubiquitin ligase complex that mono-ubiquitinates lysine  <dig> of nucleosomal histone h2a   <cit> .
the h2a-k <dig> monoubiquitination is required for the pcg-mediated in vivo gene silencing, but its role in the mechanism is not yet known  <cit> .
drosophila prc <dig> presumably possesses ubiquitin e <dig> ligase activity as well, since dring was found to co-localize with ubiquitinated h2a at the ultrabithorax  promoter  <cit> .
in drosophila and vertebrates the pcg-mediated gene silencing mechanism requires the action of both prc <dig> and prc <dig>  the apparent lack of homologs of prc <dig> components in organisms such as plants or worms and the conflicting information on histone ubiquitination in these organisms led to the speculation of alternative pcg-mediated gene silencing mechanism in which other proteins undertake prc <dig> functions  <cit> .
ubiquitination at lysine  <dig> of h2a in human was observed within the consensus peptide sequence pkkt  <cit> .
arabidopsis genome contains  <dig> histone h2a genes   <cit> , among them just hta <dig> displays conservation of the pkkt consensus sequence.
although h2a ubiquitination has not been detected in arabidopsis using western blot analysis so far  <cit> , a recent report showed the presence of ubiquitinated h2a in maize  <cit> .
on the other hand, ubiquitinated h2a has been detected in c. elegans  <cit> , indicating that some proteins may be involved in h2a ubiquitination.
the catalytic subunit of the mammalian e <dig> ubiquitin ligase complex is ring1b, but ring1a and bmi- <dig> are also required for the in vivo h2a ubiquitination  <cit> .
bmi- <dig>  ring1b and ring1a contain a ring finger domain located at the n-terminal region.
biochemical analyses showed that the n-terminal ring finger domain of ring1b is sufficient for the e <dig> ligase activity in vitro  <cit>  and that bmi- <dig> displays no detectable ubiquitin ligase activity but it greatly stimulates the e3-ligase activity of ring1b.
it was also described that in vitro ring1a can replace ring1b  <cit> ; however, the in vivo function must be distinct, as most of the ubiquitinated h2a is depleted upon lost of ring1b  <cit> .
structural analysis revealed that ring1b interacts with bmi- <dig> via their ring fingers  <cit> .
the prc <dig> ring finger proteins also share a conserved c-terminal region  <cit>  that seems to be implicated in the interaction with ph and pc  <cit> .
to gain mechanistic insight into the assembly and the enzymatic activity of the prc <dig> proteins we performed a detailed computational sequence analysis of the c-terminal region of these proteins.
our findings result in the identification of a new ubiquitin-like domain that unveiled prc <dig> ring finger-like proteins in plants and worm.
RESULTS
detection of a new ubiquitin-like domain in the prc <dig> ring finger proteins
to analyse the conserved c-terminal region of the ring finger prc <dig> proteins, we first performed blast searches  <cit>  against different sequence databases located at uniprot  <cit> , nbci  <cit> , ensembl  <cit>  and jgi  <cit> , starting from the human bmi- <dig>  alignments were generated with t-coffee  <cit>  and checked manually.
additional profile-based sequence searches were performed against the uniref <dig> and uniref  <dig> protein sequence databases  <cit>  with the defined global hidden markov models using hmmer  <cit> .
using extensive profile to sequence  and profile to profile comparisons analyses  <cit>  we identified statistically significant e-values of sequence similarity between the c-terminal conserved regions of prc <dig> ring finger proteins and the c-terminal region of the wdr48-p <dig> protein family .
the wd repeat domain  <dig> or p <dig>  family are wd <dig> repeat-containing endosomal proteins found in all eukaryotes including yeast and are present in only one copy per organism .
except for the previously described interaction between the human wdr48-p <dig> protein and a tyrosine kinase interacting protein from herpesvirus saimiri virus , the exact function of the wdr48-p <dig> family remains unknown but has been suggested to be related to endosome/lysosome traffic  <cit> .
the statistical significance of profile to profile comparisons was evaluated in terms of an e-value.
significant e-values connected all these sequences and reciprocal searches produced convergent results .
the bmi1/mel <dig> subfamily profile search finds the ring1a/1b subfamily with a  <dig> e- <dig> e-value and the global profile of the prc <dig> ring family  detected the wdr48-p <dig> and the ubl families with e-values of  <dig> e- <dig> and  <dig> e- <dig>  respectively.
the rawul hmm profile of the wdr <dig> family have less capability to detect remote protein homologies and localize the ubiquitin family with poor statistical significance , although rawul domain is more conserved inside the wdr <dig> family than in prc <dig> ring family.
nevertheless, the homology of the wdr <dig> rawul domain with the ubl family is statistically supported, when using as intermediate the rawul hmm profile of prc <dig> ring family.
this result defines a new conserved domain present in the prc <dig> ring finger and wdr <dig> families.
in all of those proteins the domain is always observed in a c-terminal location , suggesting that this is somehow important for its function.
statistically significant sequence similarity offered by profile-profile comparisons linked this domain with ubiquitin superfamily .
ubiquitin  is a highly conserved  <dig> amino acid residue protein found in all eukaryotic cells and whose sequence is extremely well conserved from protozoa to vertebrates  <cit> .
the overall topology of the ubq-like fold is β <dig>  β <dig>  α <dig>  β <dig>  β <dig>  α <dig>  β <dig> ; however, there may be deviations from this common core  <cit> .
although those e-values are significant per se, we performed three additional analyses.
first, secondary structure predictions were performed independently for the conserved c-terminal regions of prc <dig> ring finger and wdr48-p <dig> families  <cit>  .
these predictions showed good agreement with the ubq and ubq-like structures .
second, to investigate whether fold recognition analysis generated consistent results, we submitted the ubq-like domain of the human bmi- <dig> protein  to an independent fold assignment software .
mgenthreader consistently recognised only ubq-like proteins as possible templates .
this consistency in the threading output additionally supports the relationship between ubq and the conserved prc <dig> ring c-terminal domain.
third, we generated a structural model  of the human bmi- <dig> putative ubq-like domain based on alignments including ubiquitin superfamily sequences of known structure .
the model was evaluated using statistical mean force potential and found to have a z-score of - <dig> , in the upper range of good model for this length  <cit> , strengthening the assignment of the ubq fold.
despite the low sequence identity between this domain and the ubq , the hydrophobic core and key secondary structural elements of the ubq fold are conserved   <cit> .
however, the prc <dig> ring finger family displays a higher degree of sequence variability in the c-terminal ubq-like domain than the wdr48-p <dig> family.
the domain secondary structural elements are rich in charged residues and are separated by loops of variable length , which are likely to be the reasons why this domain escaped detection until now.
the fact that the domain shows insertions of variable length is not in contradiction with a stable fold  <cit> ; even more, small folds, such as ubiquitin  <cit> , immunoglobulin  <cit>  or sbds  <cit> , share a high degree of structural conservation in a scenario of high sequence divergence and long loop insertions.
the most characteristic features of this domain is the α <dig> that is a bit larger in its c-terminus compared to classical ubq-like proteins and contains a conserved pair of thr/leu that might have a structural role in capping the alpha helix .
preliminary structural characterization of the c-terminal region of ring1b showed that this domain folds independently and is a combination of alpha-helix and beta-sheet secondary structures  <cit> , which is in agreement with our analysis.
in summary, considering the profile-profile comparisons e-values , secondary structure predictions , fold assignment detection and the results of the homology model building and evaluation , we are confident that this domain is a new member of the ubq superfamily.
since we found this new ubq-like domain associated to two different domain architectures, ring-finger domain  and wd <dig> repeats , we named it rawul  domain.
the ubiquitin related proteins fall into two separate classes  <cit> .
ubq-like proteins  that function as modifiers in a manner analogous to that of ubiquitin and exist either in a free form or attached covalently to other proteins by their c-termini, and the ubiquitin-domain proteins  that bear domains related to ubiquitin but are otherwise unrelated in sequence to each other .
in contrast to the ubl modifiers, the udps are not conjugated to other proteins.
rawul domain lacks the characteristic c-terminal diglycine motif required for enzymatic conjugation of the ubq domain to an acceptor lysine.
neither are conserved the three most common acceptor lysine residues involved in polyubiquitination  as k <dig>  k <dig>  and k63).
hence, it appears unlikely that this domain functions as a traditional ubl modifier.
prc <dig> ring finger and wdr48-p <dig> proteins therefore could be included in the group of udps.
there are two classes of udps as well  <cit>  differentiated by their location and sequence similarity to ubiquitin of their ubq-like domains.
the ubq-like domain  proteins have the ubq-like domain located at or close to the n-terminus of the protein and defined by a stretch of 45– <dig> residues with significant sequence homology to ubiquitin, while the ubiquitin regulatory x  domain proteins have the ubq-like domain at the c-terminus consisting of ~ <dig> amino acids that shares common secondary structure organization with ubiquitin despite the lack of significant sequence homology  <cit> .
the ubd proteins have been shown to bind to ubiquitin-interacting motifs , like parkin protein.
defective parkin protein is responsible for a common familial form of parkinson's disease.
parkin encodes an e <dig> ubiquitin ligase that contains two ring finger domains at its c-terminus and one ubq-like domain at its n-terminus  <cit> .
binding of the parkin ubiquitin-like domain to the eps <dig> uim is required for parkin-mediated eps <dig> ubiquitination  <cit> .
conversely, only very few ubx domain proteins have been studied in detail and no general function for the ubx domain has yet emerged.
most of the ubx domain proteins identified so far can be grouped into four evolutionary conserved protein families represented by the human fas-associated factor- <dig> , p <dig>  y33k, and rep <dig> proteins  <cit> .
recent reports showed that the ubx domains may act as general binding modules for p <dig> and/or p <dig> homologs, possibly representing a first common role for ubx domains  <cit> .
rawul domain seems to fall into the ubx domain protein family since it is located at the c-terminus, comprise about  <dig> residues and the conserved hydrophobic core of the ubq fold.
however, as far as we know, ubx domains have not been found together with a ring finger domain or a wd <dig> repeat domain.
in the case of the poorly characterized wdr48-p <dig> family  <cit> , the association of wd <dig> repeats and rawul domain could point out to a e3-ligase related function, since wd <dig> repeats containing proteins are commonly implicated in protein-protein interaction  <cit>  and these repeats have been recently described as key elements in e <dig> ubiquitin ligase complexes  <cit> .
on the other side, our analysis revealed that the combination of a ring finger domain at the n-terminus and a rawul domain at the c-terminus is a key feature to define the prc <dig> ring finger protein family, since no other protein presents this domain architecture.
the ring finger domains of the ring  and bmi <dig> proteins are responsible for the e <dig> ubiquitin ligase activity  <cit> , whereas the region corresponding to the rawul domain has been shown to mediate protein-protein interaction with pc and ph  <cit> .
the identification of an ubq-like domain in this region raise the possibility that these interactions could be establish through a yet uncharacterised uim present in pc and ph.
however, due to the structural similarities between the rawul domain and ubiquitin, it is also possible that rawul could act as an auto-inhibitory domain, by direct competition with the substrate, regulating specificities and/or ubiquitination capabilities of the e <dig> ligase complex, as previously suggested for ubq-like domains fused to ubiquitin-specific proteases  <cit> , or as a bridge, linking the proteasome and histone post-translational modifications, as suggested for the ubq-like domain in the ubiquitin plant homeodomain ring finger  protein family  <cit> .
identification of plant and worm prc <dig> ring finger protein orthologs
in the course of characterizing the prc <dig> ring finger proteins, we identified a set of proteins in arabidopsis, oryza sativa, vitis vinifera and worms that share the prc <dig> ring finger domain architecture, an n-terminal ring finger domain and a rawul domain at their c-terminal region .
the preservation of domain architecture among these proteins indicates that these plant and worm proteins are potential orthologs of animal prc <dig> ring finger proteins.
the ring finger domain is highly abundant in plant proteins, with as many as  <dig> predicted ring finger-containing proteins in the arabidopsis genome  <cit> .
the large number of potential ring finger proteins in arabidopsis suggests that target specific ubiquitination plays an important role in protein regulation in plants.
however, the combination of the ring finger domain and the rawul domain, which is a specific feature of the prc <dig> ring finger proteins, is found in only a few plant proteins.
in arabidopsis, there are four prc <dig> ring finger genes, at1g <dig>  at5g <dig>  at2g <dig> and at3g <dig> that codify the predicted proteins q0wx <dig>  estq9fkw <dig>  q94ay <dig> and q9ls <dig>  respectively.
there are full length cdnas for these four genes with one open reading frame predicted for each  database  <cit> .
online microarray expression data proof that these genes are expressed  <cit> .
outside of these two domains, the plant proteins have diverged significantly from their putative animal counterparts , but usually pcg protein orthologs do not display sequence homology outside the conserved domains, not only in evolutionary distant organisms but also among paralogs in a given organism  <cit> .
taking into account the fact that convergent evolution of domain architectures is rare  <cit> , the common domain architecture presented by the plants and animal prc <dig> ring finger proteins  suggest a common evolutionary origin, raising the possibility that these plant proteins could be the prc <dig> ring finger orthologs, in turn implying that the prc1-mediated histone h2a ubiquitination might also take place in plant.
plants were thought to have no homologs of prc <dig> components.
however, two recent reports  shows that arabidopsis like heterochromatin protein <dig>  is functionally similar to pc, a subunit of prc <dig> that recognizes and binds the h3k <dig> marks created by prc <dig>  similarly, prc <dig> ring finger proteins were thought to be missing in plants; however, our analysis revealed the presence of putative orthologs to these proteins.
all together these data suggest that prc <dig> is also conserved in plants, in agreement with our analysis.
based on sequence similarity and biochemical data, two groups of prc <dig> ring finger proteins have been defined  <cit> , the bmi <dig> subfamily that includes vertebrate bmi <dig> and mel <dig> and drosophila psc and suppressor  <dig> of zeste 2), and the ring subfamily that includes vertebrate ring1a, ring1b and drosophila dring.
in order to identify potential orthologous relationships between these plant and animal homologous genes we conducted a phylogenetic analysis of this family.
due to the low degree of sequence conservation in the c-terminal domain rawul , we add the n-terminal ring domain, which is highly conserved between plants and animals , to the phylogenetic analysis of the prc <dig> ring family.
the high confidence bootstrap value  at the connecting node  indicates that both subfamilies  are clearly present in plants .
for instance, arabidopsis q0wx <dig> and estq9fkw <dig> proteins are part of the ring1a/1b subfamily, while q94ay <dig> and q9ls <dig> proteins are the orthologs of the bmi1/mel <dig> subfamily.
the identification of orthologous relationships between the two groups including both plant and animal proteins suggests an early gene duplication event, giving rise to bmi1/mel <dig> and ring1a/1b subfamilies prior to the plant-animal divergence.
the plant and animal proteins therefore could share a common biochemical function.
similarly, lower metazoans, such as nematodes, are also thought to lack prc <dig> homologs.
in c. elegans, pcg-mediated gene repression has been proposed to require a prc2-like complex  <cit>  and two novel proteins, suppressor of pal- <dig>  and sop-2-related protein  <dig>   <cit> .
despite the lack of obvious sequence similarity, several conserved properties between prc <dig> and the putative sop-2/sor- <dig> complex suggested a conservation of the mechanism  <cit> .
however, these proteins are not found in other organisms, including its closest related c. briggsae  <cit> , weakening the case for an alternative prc1-like complex.
in addition, ubiquitinated h2a has been detected by western blot analysis in c. elegans  <cit> , indicating that some proteins may be involved in h2a ubiquitination.
interestingly, we found one prc <dig> ring finger homolog in c. elegans and in c. briggsae .
the function of this prc <dig> ring finger homolog is so far unknown.
however, the conservation of the protein architecture, an n-terminal ring finger and a c-terminal rawul domain, suggest that the function of this protein could be related to histone ubiquitination.
CONCLUSIONS
we detected statistically significant sequence similarity between the ubiquitin superfamily and the conserved c-terminal regions of prc <dig> ring and wdr48-p <dig> families, defining a new ubq-like domain, the rawul domain.
the identification of an ubq-like domain in the prc <dig> ring family offers new experimental approaches aimed at elucidating their roles in important cellular processes, such us, stem cell self-renewal and cancer.
in addition, characterizing the rawul domain allowed us to identify putative prc <dig> ring finger proteins that were thought to be missing in the plants and worms.
the possibility that a prc1-like complex is also involved in pcg-mediated gene silencing mechanism in these organisms is intriguing and opens new avenues in pcg investigation.

 polycomb group proteins are epigenetic gene regulators implicated in important cellular and developmental processes. in animals, pcg proteins were primarily known for their role in maintaining cell identity during the establishment of the body plan, but recently they have also been implicated in other important processes like stem cell self-renewal and cancer. in plants, the pcg proteins are involved in the repression of flowering during vegetative development, seed development, and in the vernalization response. hence, pcg proteins play an essential role in the proper development of multicellular organisms. biochemical and genetic characterizations of pcg proteins have revealed that they exist in distinct multi-protein complexes, of which the two best characterized are the pcg repressive complex and prc the core components of prc are conserved throughout higher eukaryotes. the complex mediates histone h methylation at lysine. similar complexes have been found in caenorhabditis elegans, vertebrates and plants. unlike prc prc1-like complexes have only been described in drosophila and vertebrates. drosophila prc core complex contains posterior sex combs, polyhomeotic, drosophila ring finger protein or sex combs extra and polycomb. the complex can inhibit in vitro both chromatin remodeling by the human switch/sucrose nonfermentable complex and transcription of a chromatin template by rna polymerase ii. the human prc complex is composed of b lymphoma mo-mlv insertion region homolog, ph pc and the ring finger proteins, homologs of drosophila psc, ph, pc, and dring, respectively. human bmi ring1a and ring1b proteins form an e ubiquitin ligase complex that mono-ubiquitinates lysine of nucleosomal histone h2a. the h2a-k monoubiquitination is required for the pcg-mediated in vivo gene silencing, but its role in the mechanism is not yet known. drosophila prc presumably possesses ubiquitin e ligase activity as well, since dring was found to co-localize with ubiquitinated h2a at the ultrabithorax promoter. in drosophila and vertebrates the pcg-mediated gene silencing mechanism requires the action of both prc and prc the apparent lack of homologs of prc components in organisms such as plants or worms and the conflicting information on histone ubiquitination in these organisms led to the speculation of alternative pcg-mediated gene silencing mechanism in which other proteins undertake prc functions. ubiquitination at lysine of h2a in human was observed within the consensus peptide sequence pkkt. arabidopsis genome contains histone h2a genes, among them just hta displays conservation of the pkkt consensus sequence. although h2a ubiquitination has not been detected in arabidopsis using western blot analysis so far, a recent report showed the presence of ubiquitinated h2a in maize. on the other hand, ubiquitinated h2a has been detected in c. elegans, indicating that some proteins may be involved in h2a ubiquitination. the catalytic subunit of the mammalian e ubiquitin ligase complex is ring1b, but ring1a and bmi- are also required for the in vivo h2a ubiquitination. bmi- ring1b and ring1a contain a ring finger domain located at the n-terminal region. biochemical analyses showed that the n-terminal ring finger domain of ring1b is sufficient for the e ligase activity in vitro and that bmi- displays no detectable ubiquitin ligase activity but it greatly stimulates the e3-ligase activity of ring1b. it was also described that in vitro ring1a can replace ring1b; however, the in vivo function must be distinct, as most of the ubiquitinated h2a is depleted upon lost of ring1b. structural analysis revealed that ring1b interacts with bmi- via their ring fingers. the prc ring finger proteins also share a conserved c-terminal region that seems to be implicated in the interaction with ph and pc. to gain mechanistic insight into the assembly and the enzymatic activity of the prc proteins we performed a detailed computational sequence analysis of the c-terminal region of these proteins. our findings result in the identification of a new ubiquitin-like domain that unveiled prc ring finger-like proteins in plants and worm.  detection of a new ubiquitin-like domain in the prc ring finger proteins to analyse the conserved c-terminal region of the ring finger prc proteins, we first performed blast searches against different sequence databases located at uniprot, nbci, ensembl and jgi, starting from the human bmi- alignments were generated with t-coffee and checked manually. additional profile-based sequence searches were performed against the uniref and uniref protein sequence databases with the defined global hidden markov models using hmmer. using extensive profile to sequence and profile to profile comparisons analyses we identified statistically significant e-values of sequence similarity between the c-terminal conserved regions of prc ring finger proteins and the c-terminal region of the wdr48-p protein family. the wd repeat domain or p family are wd repeat-containing endosomal proteins found in all eukaryotes including yeast and are present in only one copy per organism. except for the previously described interaction between the human wdr48-p protein and a tyrosine kinase interacting protein from herpesvirus saimiri virus, the exact function of the wdr48-p family remains unknown but has been suggested to be related to endosome/lysosome traffic. the statistical significance of profile to profile comparisons was evaluated in terms of an e-value. significant e-values connected all these sequences and reciprocal searches produced convergent results. the bmi1/mel subfamily profile search finds the ring1a/1b subfamily with a e- e-value and the global profile of the prc ring family detected the wdr48-p and the ubl families with e-values of e- and e- respectively. the rawul hmm profile of the wdr family have less capability to detect remote protein homologies and localize the ubiquitin family with poor statistical significance, although rawul domain is more conserved inside the wdr family than in prc ring family. nevertheless, the homology of the wdr rawul domain with the ubl family is statistically supported, when using as intermediate the rawul hmm profile of prc ring family. this result defines a new conserved domain present in the prc ring finger and wdr families. in all of those proteins the domain is always observed in a c-terminal location, suggesting that this is somehow important for its function. statistically significant sequence similarity offered by profile-profile comparisons linked this domain with ubiquitin superfamily. ubiquitin is a highly conserved amino acid residue protein found in all eukaryotic cells and whose sequence is extremely well conserved from protozoa to vertebrates. the overall topology of the ubq-like fold is β β α β β α β; however, there may be deviations from this common core. although those e-values are significant per se, we performed three additional analyses. first, secondary structure predictions were performed independently for the conserved c-terminal regions of prc ring finger and wdr48-p families. these predictions showed good agreement with the ubq and ubq-like structures. second, to investigate whether fold recognition analysis generated consistent results, we submitted the ubq-like domain of the human bmi- protein to an independent fold assignment software. mgenthreader consistently recognised only ubq-like proteins as possible templates. this consistency in the threading output additionally supports the relationship between ubq and the conserved prc ring c-terminal domain. third, we generated a structural model of the human bmi- putative ubq-like domain based on alignments including ubiquitin superfamily sequences of known structure. the model was evaluated using statistical mean force potential and found to have a z-score of -, in the upper range of good model for this length, strengthening the assignment of the ubq fold. despite the low sequence identity between this domain and the ubq, the hydrophobic core and key secondary structural elements of the ubq fold are conserved. however, the prc ring finger family displays a higher degree of sequence variability in the c-terminal ubq-like domain than the wdr48-p family. the domain secondary structural elements are rich in charged residues and are separated by loops of variable length, which are likely to be the reasons why this domain escaped detection until now. the fact that the domain shows insertions of variable length is not in contradiction with a stable fold; even more, small folds, such as ubiquitin, immunoglobulin or sbds, share a high degree of structural conservation in a scenario of high sequence divergence and long loop insertions. the most characteristic features of this domain is the α that is a bit larger in its c-terminus compared to classical ubq-like proteins and contains a conserved pair of thr/leu that might have a structural role in capping the alpha helix. preliminary structural characterization of the c-terminal region of ring1b showed that this domain folds independently and is a combination of alpha-helix and beta-sheet secondary structures, which is in agreement with our analysis. in summary, considering the profile-profile comparisons e-values, secondary structure predictions, fold assignment detection and the results of the homology model building and evaluation, we are confident that this domain is a new member of the ubq superfamily. since we found this new ubq-like domain associated to two different domain architectures, ring-finger domain and wd repeats, we named it rawul domain. the ubiquitin related proteins fall into two separate classes. ubq-like proteins that function as modifiers in a manner analogous to that of ubiquitin and exist either in a free form or attached covalently to other proteins by their c-termini, and the ubiquitin-domain proteins that bear domains related to ubiquitin but are otherwise unrelated in sequence to each other. in contrast to the ubl modifiers, the udps are not conjugated to other proteins. rawul domain lacks the characteristic c-terminal diglycine motif required for enzymatic conjugation of the ubq domain to an acceptor lysine. neither are conserved the three most common acceptor lysine residues involved in polyubiquitination as k k and k63). hence, it appears unlikely that this domain functions as a traditional ubl modifier. prc ring finger and wdr48-p proteins therefore could be included in the group of udps. there are two classes of udps as well differentiated by their location and sequence similarity to ubiquitin of their ubq-like domains. the ubq-like domain proteins have the ubq-like domain located at or close to the n-terminus of the protein and defined by a stretch of 45– residues with significant sequence homology to ubiquitin, while the ubiquitin regulatory x domain proteins have the ubq-like domain at the c-terminus consisting of ~ amino acids that shares common secondary structure organization with ubiquitin despite the lack of significant sequence homology. the ubd proteins have been shown to bind to ubiquitin-interacting motifs, like parkin protein. defective parkin protein is responsible for a common familial form of parkinson's disease. parkin encodes an e ubiquitin ligase that contains two ring finger domains at its c-terminus and one ubq-like domain at its n-terminus. binding of the parkin ubiquitin-like domain to the eps uim is required for parkin-mediated eps ubiquitination. conversely, only very few ubx domain proteins have been studied in detail and no general function for the ubx domain has yet emerged. most of the ubx domain proteins identified so far can be grouped into four evolutionary conserved protein families represented by the human fas-associated factor-, p y33k, and rep proteins. recent reports showed that the ubx domains may act as general binding modules for p and/or p homologs, possibly representing a first common role for ubx domains. rawul domain seems to fall into the ubx domain protein family since it is located at the c-terminus, comprise about residues and the conserved hydrophobic core of the ubq fold. however, as far as we know, ubx domains have not been found together with a ring finger domain or a wd repeat domain. in the case of the poorly characterized wdr48-p family, the association of wd repeats and rawul domain could point out to a e3-ligase related function, since wd repeats containing proteins are commonly implicated in protein-protein interaction and these repeats have been recently described as key elements in e ubiquitin ligase complexes. on the other side, our analysis revealed that the combination of a ring finger domain at the n-terminus and a rawul domain at the c-terminus is a key feature to define the prc ring finger protein family, since no other protein presents this domain architecture. the ring finger domains of the ring and bmi proteins are responsible for the e ubiquitin ligase activity, whereas the region corresponding to the rawul domain has been shown to mediate protein-protein interaction with pc and ph. the identification of an ubq-like domain in this region raise the possibility that these interactions could be establish through a yet uncharacterised uim present in pc and ph. however, due to the structural similarities between the rawul domain and ubiquitin, it is also possible that rawul could act as an auto-inhibitory domain, by direct competition with the substrate, regulating specificities and/or ubiquitination capabilities of the e ligase complex, as previously suggested for ubq-like domains fused to ubiquitin-specific proteases, or as a bridge, linking the proteasome and histone post-translational modifications, as suggested for the ubq-like domain in the ubiquitin plant homeodomain ring finger protein family. identification of plant and worm prc ring finger protein orthologs in the course of characterizing the prc ring finger proteins, we identified a set of proteins in arabidopsis, oryza sativa, vitis vinifera and worms that share the prc ring finger domain architecture, an n-terminal ring finger domain and a rawul domain at their c-terminal region. the preservation of domain architecture among these proteins indicates that these plant and worm proteins are potential orthologs of animal prc ring finger proteins. the ring finger domain is highly abundant in plant proteins, with as many as predicted ring finger-containing proteins in the arabidopsis genome. the large number of potential ring finger proteins in arabidopsis suggests that target specific ubiquitination plays an important role in protein regulation in plants. however, the combination of the ring finger domain and the rawul domain, which is a specific feature of the prc ring finger proteins, is found in only a few plant proteins. in arabidopsis, there are four prc ring finger genes, at1g at5g at2g and at3g that codify the predicted proteins q0wx estq9fkw q94ay and q9ls respectively. there are full length cdnas for these four genes with one open reading frame predicted for each database. online microarray expression data proof that these genes are expressed. outside of these two domains, the plant proteins have diverged significantly from their putative animal counterparts, but usually pcg protein orthologs do not display sequence homology outside the conserved domains, not only in evolutionary distant organisms but also among paralogs in a given organism. taking into account the fact that convergent evolution of domain architectures is rare, the common domain architecture presented by the plants and animal prc ring finger proteins suggest a common evolutionary origin, raising the possibility that these plant proteins could be the prc ring finger orthologs, in turn implying that the prc1-mediated histone h2a ubiquitination might also take place in plant. plants were thought to have no homologs of prc components. however, two recent reports shows that arabidopsis like heterochromatin protein is functionally similar to pc, a subunit of prc that recognizes and binds the h3k marks created by prc similarly, prc ring finger proteins were thought to be missing in plants; however, our analysis revealed the presence of putative orthologs to these proteins. all together these data suggest that prc is also conserved in plants, in agreement with our analysis. based on sequence similarity and biochemical data, two groups of prc ring finger proteins have been defined, the bmi subfamily that includes vertebrate bmi and mel and drosophila psc and suppressor of zeste 2), and the ring subfamily that includes vertebrate ring1a, ring1b and drosophila dring. in order to identify potential orthologous relationships between these plant and animal homologous genes we conducted a phylogenetic analysis of this family. due to the low degree of sequence conservation in the c-terminal domain rawul, we add the n-terminal ring domain, which is highly conserved between plants and animals, to the phylogenetic analysis of the prc ring family. the high confidence bootstrap value at the connecting node indicates that both subfamilies are clearly present in plants. for instance, arabidopsis q0wx and estq9fkw proteins are part of the ring1a/1b subfamily, while q94ay and q9ls proteins are the orthologs of the bmi1/mel subfamily. the identification of orthologous relationships between the two groups including both plant and animal proteins suggests an early gene duplication event, giving rise to bmi1/mel and ring1a/1b subfamilies prior to the plant-animal divergence. the plant and animal proteins therefore could share a common biochemical function. similarly, lower metazoans, such as nematodes, are also thought to lack prc homologs. in c. elegans, pcg-mediated gene repression has been proposed to require a prc2-like complex and two novel proteins, suppressor of pal- and sop-2-related protein. despite the lack of obvious sequence similarity, several conserved properties between prc and the putative sop-2/sor- complex suggested a conservation of the mechanism. however, these proteins are not found in other organisms, including its closest related c. briggsae, weakening the case for an alternative prc1-like complex. in addition, ubiquitinated h2a has been detected by western blot analysis in c. elegans, indicating that some proteins may be involved in h2a ubiquitination. interestingly, we found one prc ring finger homolog in c. elegans and in c. briggsae. the function of this prc ring finger homolog is so far unknown. however, the conservation of the protein architecture, an n-terminal ring finger and a c-terminal rawul domain, suggest that the function of this protein could be related to histone ubiquitination.  we detected statistically significant sequence similarity between the ubiquitin superfamily and the conserved c-terminal regions of prc ring and wdr48-p families, defining a new ubq-like domain, the rawul domain. the identification of an ubq-like domain in the prc ring family offers new experimental approaches aimed at elucidating their roles in important cellular processes, such us, stem cell self-renewal and cancer. in addition, characterizing the rawul domain allowed us to identify putative prc ring finger proteins that were thought to be missing in the plants and worms. the possibility that a prc1-like complex is also involved in pcg-mediated gene silencing mechanism in these organisms is intriguing and opens new avenues in pcg investigation. 
the two best-characterized pcg complexes are the pcg repressive complex and that respectively possess histone 2a lysine e ubiquitin ligase and histone lysine methyltransferase activities. these prc proteins have an n-terminally located ring finger domain associated to a poorly characterized conserved c-terminal region. we obtained statistically significant evidences of sequence similarity between the c-terminal region of the prc ring finger proteins and the ubiquitin -like family proteins, thus defining a new ubq-like domain, the rawul domain. in addition, our analysis revealed the existence of plant and worm proteins that display the conserved combination of a ring finger domain at the n-terminus and a rawul domain at the c-terminus. analysis of the conserved domain architecture among prc ring finger proteins revealed the existence of long sought prc protein orthologs in these organisms, suggesting the functional conservation of prc throughout higher eukaryotes.
 polycomb group proteins are a set of chromatin-modifying proteins that play a key role in epigenetic gene regulation. the pcg proteins form large multiprotein complexes with different activities. the two best-characterized pcg complexes are the pcg repressive complex and that respectively possess histone 2a lysine e ubiquitin ligase and histone lysine methyltransferase activities. while prc2-like complexes are conserved throughout the eukaryotic kingdoms, prc1-like complexes have only been described in drosophila and vertebrates. since both complexes are required for the gene silencing mechanism in drosophila and vertebrates, how prc function is realized in organisms that apparently lack prc such as plants, is so far unknown. in vertebrates, prc includes three proteins, ring1b, ring1a, and bmi- that form an e ubiquitin ligase complex. these prc proteins have an n-terminally located ring finger domain associated to a poorly characterized conserved c-terminal region.  we obtained statistically significant evidences of sequence similarity between the c-terminal region of the prc ring finger proteins and the ubiquitin -like family proteins, thus defining a new ubq-like domain, the rawul domain. in addition, our analysis revealed the existence of plant and worm proteins that display the conserved combination of a ring finger domain at the n-terminus and a rawul domain at the c-terminus.  analysis of the conserved domain architecture among prc ring finger proteins revealed the existence of long sought prc protein orthologs in these organisms, suggesting the functional conservation of prc throughout higher eukaryotes. 