BACKGROUND
among ubiquitin-related proteins, containing at least one domain with a ubiquitin-like fold, one can distinguish ubiquitin-like modifiers  and ubiquitin domain proteins   <cit> .
ubls can be covalently attached to target proteins analogously to ubiquitin.
unlike ubiquitin, ubls mostly do not directly target proteins for degradation  <cit> , although functional links can exist.
one of the most heavily researched single domain ubls, the small ubiquitin-related modifier , is known to act on transcription factors, chromatin associated proteins, nuclear body proteins and septins  <cit> .
in contrast to ubls, udps are not conjugated to other proteins and lack the c-terminal double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers.
they are a heterogeneous class of usually multi-domain proteins, which are unrelated outside of their ubiquitin-like domain  <cit> .
in several cases, it has been demonstrated that the ubiquitin-domain within those proteins likely fulfills its cellular role by functionally mimicking ubiquitination  <cit> .
the biological relevance of non-conjugatable multi-domain proteins having a domain with clear relationship to ubls like sumo, rather than ubiquitin, is yet unknown.
here, we present a detailed sequence analysis of a family of sumo-like domain proteins  containing one or two sumo-like domains.
members of the proposed reni family act as factors in transcriptional regulation, chromatin silencing and genomic stability.
RESULTS
sequence architecture of drosophila melanogaster cg4449
during the study of the predicted nuclear subset of the drosophila proteome, we encountered the unknown  <dig> amino acids long protein cg <dig> .
initial analysis of its sequence complexity shows that the disordered n-terminal half of the protein is followed by a likely globular segment .
indeed, a compositionally biased, polar low-complexity region  spans almost the entire n-terminal  <dig> amino acids  as reported by cast   <cit>  and seg   <cit> .
the c-terminal half of cg <dig> turns out to contain an internally repeated segment identifiable with radar  <cit>  .
in an attempt to confirm this repeat, we queried the protein against the conserved domain database using rps-blast  <cit> .
thereby, we could define a similarity to sumo-like domains overlapping with the second repeat-element , while no significant hits emerged for the first of the repeat-constituents.
using profile-profile comparison, however, segment 220– <dig> is shown to possess a distant, yet significant similarity to sumo sequences and, therefore, to share the sumo fold .
in conclusion, we found that the drosophila protein cg <dig>  has a tripartite architecture: with a n-terminal lcr followed by two globular domains with a sumo-like fold .
whereas sd2's similarity to single domain sumo-like sequences can be easily detected with blast tools, the identification of sd <dig> is non-trivial .
for both sd <dig> and sd <dig>  a carboxy-terminal double-glycine motif, as it is known and necessary for the covalent attachment of sumo proteins, is missing.
this finding is remarkable as sumo proteins are discussed in the scientific community solely as polypeptides that become covalently bonded to various targets  <cit> .
here, we present cases of non-conjugatable poly-sumo fusion protein.
collecting animal nip45-related proteins characterized by two sumo-like domains
a psi-blast search started with the globular c-terminal half of cg <dig>  including the two sumo-like domains, collects a family of animal proteins with the same tripartite organization in c. briggsae , h. sapiens , m. musculus  and c. elegans .
all these proteins contain a lcr at the n-terminus followed by two sumo-like domains, the first of which has mostly diverged away beyond recognition thresholds using traditional sequence-profile searches .
the human and the mouse homologs correspond to the studied nuclear factor nip <dig>   <cit> .
all sequences and original database search results can be found at the reni homepage  <cit> .
distant nip <dig> homologs in fungi, other lower eukaryotes and plants
indications on the existence of nip <dig> homologs in lower eukaryotes and plants
a multiple sequence alignment of the globular c-terminal half of d. melanogaster cg <dig>  and the corresponding sequences derived from a. gambie, x. laevis, c. elegans, c. briggsae, m. musculus and h. sapiens  was used to generate a hidden markov model .
the protein family was enlarged using the hmmer <dig> tool  <cit>  in searches against single model organism proteomes.
these searches retrieved as best hits in the respective proteomes the likely homologs in d. discoideum  a. thaliana , o. sativa , s. pombe , s. cerevisiae , y. lipolytica , c. glabrata  and in other recently published fungi proteomes  <cit> .
the s. cerevisiae and s. pombe homologs correspond to the studied rad <dig> and esc <dig> proteins, respectively  <cit> ; the remaining proteins are uncharacterized.
these hmm-search results suggest the most likely plant and lower eukaryote orthologues to the animal nip45-like proteins.
for establishing the orthology relationship, these initial results need to be confirmed by reciprocal searches independently performed for fungal and plant proteins.
further below, we present this evidence for the homology between the c-terminal part of the proteins found in the various taxonomic groups.
confirming fungal family members in reciprocal searches
the set of fungal reni proteins can be autonomously collected using a blastp search started with the c. glabrata representative  and retrieving the best and significant hits in the proteomes of s. cerevisiae, s. pombe, k. lactis, c. albicans, y. lipolytica, d. hansenii, a. nidulans  <cit> .
the domain architecture of these fungal homologs is likely also tripartite .
it differs from the animal representatives by a longer sequence separating the two sumo-like domains , which is typically of highly helical content .
a hmm was generated from a multiple sequence alignment of the sumo-domain containing c-terminal half of the listed fungal homologs, where gap only columns replaced the compositional biased helical region between the two sumo domains.
a search with this hmm retrieved as best hits in the respective proteomes the reni proteins in m. musculus , a. thaliana , c. elegans .
confirming plant family members in reciprocal searches
potential plant reni homologs, derived in a full-length tblastn search with a. thaliana  against the tigr gene indices of barley, maize, rice, potato and soybean  <cit> , show a length of 210– <dig> aa and are thus around  <dig> aa shorter than the shortest animal homolog from worm.
the domain organization seems also to be distinct.
a  <dig> aa n-terminal, very polar region  is followed by ~ <dig> aa with predicted strong helical preference  and a clear c-terminal sumo-like domain .
there are no indications for another sumo-like domain at the n-terminal side of the ~ <dig> aa helical region.
further confirmation of the relationship between the listed reni proteins of the viridiplantae and fungi/metazoan group comes from the analysis of the reciprocal genomic best hits of a. thaliana in y. lipolytica  and h. sapiens proteomes   <cit> .
definition of the rad60-esc2-nip <dig>  protein family
we propose to name the collected group of protein sequences the reni-family after its most studied members rad <dig>  esc <dig> and nip <dig>  all representatives have a similar sequence architecture involving a n-terminal low complexity region with many polar and  charged residues and a c-terminal globular part with one  or two  sumo-like domains.
the use of a model representing the complete globular region of reni proteins was essential for the successful collection of the family.
a global hmm spanning the sd <dig> and sd <dig> domains tests for homology in the whole globular part and, correspondingly, directly collects the reni family.
in contrast, when using the c-terminal half of various reni family members as query sequence in psi-blast  <cit> , the searches are invaded by sumo proteins  before the reni family can be collected.
this means that the sd <dig> domain sequence segments of the reni group and the family of single-domain sumo proteins are not well separated in sequence space .
for this reason, the similarity of nip <dig>  esc <dig> and rad <dig> could previously only been defined transitively via the similarity of their sd <dig> segment to sumo proteins and their similar length  <cit> .
discussion
the sumo-like domains in proteins of the rad60-esc2-nip <dig>  family
while reni proteins of the fungal, metazoan and mycetozoan taxa contain two c-terminal sumo-like domains , only the second one can be clearly defined in plant representatives .
this very c-terminal sd <dig> domain, shares several features discriminating sumo proteins from other ubiquitin-like modifiers, as for example the large negative charged cluster, seen in the alignment 5– <dig> residues from the very c-terminus .
the negative surface patch formed by these residues has been suggested to shape a sumo-typical interaction surface  <cit> .
reni proteins lack conservation of the carboxy-terminal double-glycine motif required for covalent attachment of sumo to its substrates.
thus, they are likely linear non-cleavable sumo fusions, which cannot be conjugated to target proteins, and have to be classified as udps.
the sd <dig> sumo-like domain contained in fungi, metazoa and mycetozoa, has sequentially diverged away from sumo proteins, but structural prediction suggest its resemblance to sumo .
the low sequence conservation of this domain does not understate a possible functional conservation in that region, as it has been shown that the structure rather than sequence is important for the function of ubiquitin-like domains .
for example, replacing the ud of the udp rad <dig> with ubiquitin renders a functional protein variant  <cit> .
indications on the functional importance of the first sumo-like domain in reni proteins come from the two fungal representatives of the family.
the fission yeast rad60- <dig>   <cit>  and rad60- <dig>   <cit>  mutants, which are defective in the rad <dig> function of double strand break repair, contain a point-mutation within this first sumo-like domain .
the sequence alignment to human sumo- <dig>  indicates that both mutations most likely affect structurally important positions.
they align to residues within human sumo- <dig>   that have been listed by bayer et al.
<cit>  among the contacts contributing with parts of their side chain to the formation of the hydrophobic core of the fold .
in the budding yeast esc2p, the region containing the first sumo-domain sd <dig> together with a  <dig> aa low-complex n-terminal segment can be defined as a sufficient fragment supplying its function in targeted silencing   <cit> .
a graph representation of the pair-wise similarity relationship for sd <dig> and sd <dig> sequences to other known ubiquitin-like domains  illustrates that both are most closely related to sumo domains.
from our analysis of sequence similarity, we suggest that, at least, the very c-terminal sumo-like domain  in reni proteins is able to mimic sumo and potentially shares its interaction partners.
on the other hand, the available experimental data confirms the functional importance of the sumo-like domain sd <dig> preceding it.
the n-terminal polar low complexity region in proteins of the rad60-esc2-nip <dig>  family
the occurrence of a n-terminal low complexity region with an excess of polar/charged residues is a characteristic element of the reni protein architecture.
most likely, this is a conformationally flexible segment without inherent structural preference  <cit> .
the molecular function of this region remains unknown.
it should also be noted that homology considerations are not applicable to such compositionally biased regions for functional prediction.
boddy et al.
<cit>  discussed the possible existence of coiled coils in the domain architecture of rad <dig>  esc <dig> and nip <dig>  we found that the coils  <cit>  tool generates hits only in few representatives of the reni family.
they are positionally not conserved relative to the two sumo-like domains.
it is known that the coils tool produces a considerable number of false-positive hits, especially in regions with many polar/charged residues, for example, as is likely the case for a glutamic-acid-rich part in rad <dig>
experimentally verified functions of reni proteins
functional information about reni family members is restricted to the fungal esc <dig>  rad <dig> and the metazoan nip <dig> proteins.
here, unfortunately most of the existing data relates to the full-length sequences.
nevertheless, the quite divergent set of functions known for reni proteins shows considerable overlap with the established cellular roles of sumo proteins in genome replication and regulation of gene expression.
the fission yeast rad <dig> protein was shown to be essential in dna double-strand break repair, and to be critical also for normal growth  <cit> .
it physically and genetically interacts with the smc5/ <dig> complex, a complex with a housekeeping role in the genome  <cit> .
interestingly, the smc5/ <dig> complex  <cit>  also includes nse <dig>  a protein containing a zf-miz domain commonly found in e3-like sumo ligases .
in addition, rad <dig> is known to bind the replication checkpoint kinase cds <dig>  <cit> .
s. cerevisiae esc <dig>  is involved in chromatin silencing via the recruitment or stabilization of the sir  complex  <cit> .
it is known to interact with sir <dig>  a histone nad-dependent deacetylase  of the sir complex, which is well conserved from bacteria to human  <cit>  and, thus, might be an interaction partner of other reni proteins as well.
similarly to other hdacs, sir <dig> proteins are recruited to chromatin by dna-bound factors  <cit>  and act by deacetylating histones  <cit>  as well as transcription factors such as p <dig> and forkhead transcription factor   <cit> .
with respect to a possible intersection with known rad <dig> functions, it is interesting that sir <dig> is not only involved in heterochromatic gene silencing and euchromatic repression  <cit>  but also in dna double-strand break repair mediated by end-joining  <cit> .
nip <dig>  the one studied reni in metazoa, has been implied in gene regulation, where it needs its dna-binding partner nfatp for this activity  <cit> .
strikingly, the nfat family member nfat <dig> that interacts with nip <dig>  <cit>  was independently shown to be sumoylated  <cit> .
nfat <dig> sumoylation acts in nuclear retention, regulation of transcriptional activity and recruitment to nuclear sumo- <dig> bodies  <cit> .
this analysis might suggest a potential role for sumo-like nip <dig> in its complex with nfat proteins.
possible functional role of the sumo-like domains in reni proteins
there is little experimental data on the importance of the predicted sumo-like domains in reni proteins.
nevertheless, all listed functions of reni proteins conform with the known role of sumo in transcriptional regulation and the control of genome integrity  <cit> .
in the context of transcriptional repression, sumo-modification has been suggested to recruit class i and ii hdacs to promoter sites.
regarding genome stability, sumoylation in dna-repair proteins is thought to target these to dna damage foci.
the following parallels in reni proteins become obvious: 1) hdac recruitment has also been suggested for the fission yeast esc2p  <cit> .
2) mammalian nip <dig> binds to transcription factors that can also be modified by sumo  <cit> .
3) reni and sumo share a functional context in double-strand break repair and transcriptional regulation.
on the basis of functional overlaps of sumo and reni proteins, we can speculate that reni proteins act as sumo stable fusion proteins "mimicking" sumo and that they might have common interaction partners.
CONCLUSIONS
in this report, we use sequence-analytical methods to infer the homology relationships between reni family members and determine their tripartite  domain architecture.
a n-terminal polar low-complexity segment and two consecutive sumo-like domains in the c-terminal half characterize the functionally described fungal and metazoan reni proteins.
while the more c-terminal sd <dig> is easily detectable, it is the particularly divergent sd <dig> that was shown in fungi to be essential for the assayed molecular functions.
due to the likely limited sequential-  requirements, this sumo-like domain is difficult to detect and has been missed in previous analyses of individual family members.
the identification of the more n-terminal sumo-like domain sd <dig> helps rationalizing experimental findings for mutant fungal reni family members.

 among ubiquitin-related proteins, containing at least one domain with a ubiquitin-like fold, one can distinguish ubiquitin-like modifiers and ubiquitin domain proteins. ubls can be covalently attached to target proteins analogously to ubiquitin. unlike ubiquitin, ubls mostly do not directly target proteins for degradation, although functional links can exist. one of the most heavily researched single domain ubls, the small ubiquitin-related modifier, is known to act on transcription factors, chromatin associated proteins, nuclear body proteins and septins. in contrast to ubls, udps are not conjugated to other proteins and lack the c-terminal double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. they are a heterogeneous class of usually multi-domain proteins, which are unrelated outside of their ubiquitin-like domain. in several cases, it has been demonstrated that the ubiquitin-domain within those proteins likely fulfills its cellular role by functionally mimicking ubiquitination. the biological relevance of non-conjugatable multi-domain proteins having a domain with clear relationship to ubls like sumo, rather than ubiquitin, is yet unknown. here, we present a detailed sequence analysis of a family of sumo-like domain proteins containing one or two sumo-like domains. members of the proposed reni family act as factors in transcriptional regulation, chromatin silencing and genomic stability.  sequence architecture of drosophila melanogaster cg4449 during the study of the predicted nuclear subset of the drosophila proteome, we encountered the unknown amino acids long protein cg. initial analysis of its sequence complexity shows that the disordered n-terminal half of the protein is followed by a likely globular segment. indeed, a compositionally biased, polar low-complexity region spans almost the entire n-terminal amino acids as reported by cast and seg. the c-terminal half of cg turns out to contain an internally repeated segment identifiable with radar. in an attempt to confirm this repeat, we queried the protein against the conserved domain database using rps-blast. thereby, we could define a similarity to sumo-like domains overlapping with the second repeat-element, while no significant hits emerged for the first of the repeat-constituents. using profile-profile comparison, however, segment 220– is shown to possess a distant, yet significant similarity to sumo sequences and, therefore, to share the sumo fold. in conclusion, we found that the drosophila protein cg has a tripartite architecture: with a n-terminal lcr followed by two globular domains with a sumo-like fold. whereas sd2's similarity to single domain sumo-like sequences can be easily detected with blast tools, the identification of sd is non-trivial. for both sd and sd a carboxy-terminal double-glycine motif, as it is known and necessary for the covalent attachment of sumo proteins, is missing. this finding is remarkable as sumo proteins are discussed in the scientific community solely as polypeptides that become covalently bonded to various targets. here, we present cases of non-conjugatable poly-sumo fusion protein. collecting animal nip45-related proteins characterized by two sumo-like domains a psi-blast search started with the globular c-terminal half of cg including the two sumo-like domains, collects a family of animal proteins with the same tripartite organization in c. briggsae, h. sapiens, m. musculus and c. elegans. all these proteins contain a lcr at the n-terminus followed by two sumo-like domains, the first of which has mostly diverged away beyond recognition thresholds using traditional sequence-profile searches. the human and the mouse homologs correspond to the studied nuclear factor nip. all sequences and original database search results can be found at the reni homepage. distant nip homologs in fungi, other lower eukaryotes and plants indications on the existence of nip homologs in lower eukaryotes and plants a multiple sequence alignment of the globular c-terminal half of d. melanogaster cg and the corresponding sequences derived from a. gambie, x. laevis, c. elegans, c. briggsae, m. musculus and h. sapiens was used to generate a hidden markov model. the protein family was enlarged using the hmmer tool in searches against single model organism proteomes. these searches retrieved as best hits in the respective proteomes the likely homologs in d. discoideum a. thaliana, o. sativa, s. pombe, s. cerevisiae, y. lipolytica, c. glabrata and in other recently published fungi proteomes. the s. cerevisiae and s. pombe homologs correspond to the studied rad and esc proteins, respectively; the remaining proteins are uncharacterized. these hmm-search results suggest the most likely plant and lower eukaryote orthologues to the animal nip45-like proteins. for establishing the orthology relationship, these initial results need to be confirmed by reciprocal searches independently performed for fungal and plant proteins. further below, we present this evidence for the homology between the c-terminal part of the proteins found in the various taxonomic groups. confirming fungal family members in reciprocal searches the set of fungal reni proteins can be autonomously collected using a blastp search started with the c. glabrata representative and retrieving the best and significant hits in the proteomes of s. cerevisiae, s. pombe, k. lactis, c. albicans, y. lipolytica, d. hansenii, a. nidulans. the domain architecture of these fungal homologs is likely also tripartite. it differs from the animal representatives by a longer sequence separating the two sumo-like domains, which is typically of highly helical content. a hmm was generated from a multiple sequence alignment of the sumo-domain containing c-terminal half of the listed fungal homologs, where gap only columns replaced the compositional biased helical region between the two sumo domains. a search with this hmm retrieved as best hits in the respective proteomes the reni proteins in m. musculus, a. thaliana, c. elegans. confirming plant family members in reciprocal searches potential plant reni homologs, derived in a full-length tblastn search with a. thaliana against the tigr gene indices of barley, maize, rice, potato and soybean, show a length of 210– aa and are thus around aa shorter than the shortest animal homolog from worm. the domain organization seems also to be distinct. a aa n-terminal, very polar region is followed by ~ aa with predicted strong helical preference and a clear c-terminal sumo-like domain. there are no indications for another sumo-like domain at the n-terminal side of the ~ aa helical region. further confirmation of the relationship between the listed reni proteins of the viridiplantae and fungi/metazoan group comes from the analysis of the reciprocal genomic best hits of a. thaliana in y. lipolytica and h. sapiens proteomes. definition of the rad60-esc2-nip protein family we propose to name the collected group of protein sequences the reni-family after its most studied members rad esc and nip all representatives have a similar sequence architecture involving a n-terminal low complexity region with many polar and charged residues and a c-terminal globular part with one or two sumo-like domains. the use of a model representing the complete globular region of reni proteins was essential for the successful collection of the family. a global hmm spanning the sd and sd domains tests for homology in the whole globular part and, correspondingly, directly collects the reni family. in contrast, when using the c-terminal half of various reni family members as query sequence in psi-blast, the searches are invaded by sumo proteins before the reni family can be collected. this means that the sd domain sequence segments of the reni group and the family of single-domain sumo proteins are not well separated in sequence space. for this reason, the similarity of nip esc and rad could previously only been defined transitively via the similarity of their sd segment to sumo proteins and their similar length. discussion the sumo-like domains in proteins of the rad60-esc2-nip family while reni proteins of the fungal, metazoan and mycetozoan taxa contain two c-terminal sumo-like domains, only the second one can be clearly defined in plant representatives. this very c-terminal sd domain, shares several features discriminating sumo proteins from other ubiquitin-like modifiers, as for example the large negative charged cluster, seen in the alignment 5– residues from the very c-terminus. the negative surface patch formed by these residues has been suggested to shape a sumo-typical interaction surface. reni proteins lack conservation of the carboxy-terminal double-glycine motif required for covalent attachment of sumo to its substrates. thus, they are likely linear non-cleavable sumo fusions, which cannot be conjugated to target proteins, and have to be classified as udps. the sd sumo-like domain contained in fungi, metazoa and mycetozoa, has sequentially diverged away from sumo proteins, but structural prediction suggest its resemblance to sumo. the low sequence conservation of this domain does not understate a possible functional conservation in that region, as it has been shown that the structure rather than sequence is important for the function of ubiquitin-like domains. for example, replacing the ud of the udp rad with ubiquitin renders a functional protein variant. indications on the functional importance of the first sumo-like domain in reni proteins come from the two fungal representatives of the family. the fission yeast rad60- and rad60- mutants, which are defective in the rad function of double strand break repair, contain a point-mutation within this first sumo-like domain. the sequence alignment to human sumo- indicates that both mutations most likely affect structurally important positions. they align to residues within human sumo- that have been listed by bayer et al. among the contacts contributing with parts of their side chain to the formation of the hydrophobic core of the fold. in the budding yeast esc2p, the region containing the first sumo-domain sd together with a aa low-complex n-terminal segment can be defined as a sufficient fragment supplying its function in targeted silencing. a graph representation of the pair-wise similarity relationship for sd and sd sequences to other known ubiquitin-like domains illustrates that both are most closely related to sumo domains. from our analysis of sequence similarity, we suggest that, at least, the very c-terminal sumo-like domain in reni proteins is able to mimic sumo and potentially shares its interaction partners. on the other hand, the available experimental data confirms the functional importance of the sumo-like domain sd preceding it. the n-terminal polar low complexity region in proteins of the rad60-esc2-nip family the occurrence of a n-terminal low complexity region with an excess of polar/charged residues is a characteristic element of the reni protein architecture. most likely, this is a conformationally flexible segment without inherent structural preference. the molecular function of this region remains unknown. it should also be noted that homology considerations are not applicable to such compositionally biased regions for functional prediction. boddy et al. discussed the possible existence of coiled coils in the domain architecture of rad esc and nip we found that the coils tool generates hits only in few representatives of the reni family. they are positionally not conserved relative to the two sumo-like domains. it is known that the coils tool produces a considerable number of false-positive hits, especially in regions with many polar/charged residues, for example, as is likely the case for a glutamic-acid-rich part in rad experimentally verified functions of reni proteins functional information about reni family members is restricted to the fungal esc rad and the metazoan nip proteins. here, unfortunately most of the existing data relates to the full-length sequences. nevertheless, the quite divergent set of functions known for reni proteins shows considerable overlap with the established cellular roles of sumo proteins in genome replication and regulation of gene expression. the fission yeast rad protein was shown to be essential in dna double-strand break repair, and to be critical also for normal growth. it physically and genetically interacts with the smc5/ complex, a complex with a housekeeping role in the genome. interestingly, the smc5/ complex also includes nse a protein containing a zf-miz domain commonly found in e3-like sumo ligases. in addition, rad is known to bind the replication checkpoint kinase cds. s. cerevisiae esc is involved in chromatin silencing via the recruitment or stabilization of the sir complex. it is known to interact with sir a histone nad-dependent deacetylase of the sir complex, which is well conserved from bacteria to human and, thus, might be an interaction partner of other reni proteins as well. similarly to other hdacs, sir proteins are recruited to chromatin by dna-bound factors and act by deacetylating histones as well as transcription factors such as p and forkhead transcription factor. with respect to a possible intersection with known rad functions, it is interesting that sir is not only involved in heterochromatic gene silencing and euchromatic repression but also in dna double-strand break repair mediated by end-joining. nip the one studied reni in metazoa, has been implied in gene regulation, where it needs its dna-binding partner nfatp for this activity. strikingly, the nfat family member nfat that interacts with nip was independently shown to be sumoylated. nfat sumoylation acts in nuclear retention, regulation of transcriptional activity and recruitment to nuclear sumo- bodies. this analysis might suggest a potential role for sumo-like nip in its complex with nfat proteins. possible functional role of the sumo-like domains in reni proteins there is little experimental data on the importance of the predicted sumo-like domains in reni proteins. nevertheless, all listed functions of reni proteins conform with the known role of sumo in transcriptional regulation and the control of genome integrity. in the context of transcriptional repression, sumo-modification has been suggested to recruit class i and ii hdacs to promoter sites. regarding genome stability, sumoylation in dna-repair proteins is thought to target these to dna damage foci. the following parallels in reni proteins become obvious: 1) hdac recruitment has also been suggested for the fission yeast esc2p. 2) mammalian nip binds to transcription factors that can also be modified by sumo. 3) reni and sumo share a functional context in double-strand break repair and transcriptional regulation. on the basis of functional overlaps of sumo and reni proteins, we can speculate that reni proteins act as sumo stable fusion proteins "mimicking" sumo and that they might have common interaction partners.  in this report, we use sequence-analytical methods to infer the homology relationships between reni family members and determine their tripartite domain architecture. a n-terminal polar low-complexity segment and two consecutive sumo-like domains in the c-terminal half characterize the functionally described fungal and metazoan reni proteins. while the more c-terminal sd is easily detectable, it is the particularly divergent sd that was shown in fungi to be essential for the assayed molecular functions. due to the likely limited sequential- requirements, this sumo-like domain is difficult to detect and has been missed in previous analyses of individual family members. the identification of the more n-terminal sumo-like domain sd helps rationalizing experimental findings for mutant fungal reni family members. 
we describe the reni family of sumo-like domain proteins with the unique feature of typically containing two carboxy-terminal sumo-like domains. we suggest that the sumo stable fusion proteins of the reni family, which we introduce in this work, might mimic sumo and share its interaction motifs. recent findings point at the importance of sumo-mediated histone nad-dependent deacetylase recruitment in transcriptional regulation. here, we present the exciting possibility that sumo domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. but the specific molecular effects of sumo modification on many target proteins remain to be elucidated. till now, sumo-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. different proteins of the novel family are known to interact directly with histone nad-dependent deacetylases, structural maintenance of chromosomes proteins, and transcription factors. transcription factors and chromatin-associated proteins and in the recruitment of hdac-activity.
 post-translational modification by small ubiquitin-like modifiers has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. but the specific molecular effects of sumo modification on many target proteins remain to be elucidated. recent findings point at the importance of sumo-mediated histone nad-dependent deacetylase recruitment in transcriptional regulation.  we describe the reni family of sumo-like domain proteins with the unique feature of typically containing two carboxy-terminal sumo-like domains. using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast rad esc and mouse nip. different proteins of the novel family are known to interact directly with histone nad-dependent deacetylases, structural maintenance of chromosomes proteins, and transcription factors. in particular, the highly non-trivial designation of the first of the two successive sumo-domains in non-plant reni provides a rationale for previously published functionally impaired mutant variants.  till now, sumo-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. here, we present the exciting possibility that sumo domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. we suggest that the sumo stable fusion proteins of the reni family, which we introduce in this work, might mimic sumo and share its interaction motifs. this presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of hdac-activity. 