BACKGROUND
the nucleus not only houses the genetic material but also administers its transcription.
morphologically defined intra-nuclear compartments or bodies are non-randomly interspersed amongst chromosomes.
preliminary evidence indicates that these compartments are spatially and functionally organized, and play important regulatory roles.
for example, nucleoli are primarily concerned with ribosomal biogenesis at sites of ribosomal genes, nuclear speckles accumulate pre-mrna splicing factors at actively transcribed genes, and nuclear pore complexes sit in the nuclear membrane to control import and export of material.
other compartments, such as cajal and pml nuclear bodies, appear to be involved in a much broader range of functions.
the modular nature of the nuclear milieu and the fluid means of molecular transport enable several levels of regulation.
for example, pml nuclear bodies assemble in response to sumoylation .
they also retain and release regulatory proteins in response to cellular stress .
similarly, the nucleolus sequesters regulatory proteins for timely release .
at a more general level, nuclear pores regulate protein access to the nucleus  <cit> .
this paper paves the way for the computational mapping of the role of nuclear architecture in critical regulatory processes.
it taps recent experimental data and the growing knowledge of nuclear architecture to portray the function and the protein complement of intra-nuclear compartments.
it is important to note that proteins and rna do not constitutively and exclusively localize to a single nuclear compartment.
data sets and models need to account for this biochemically fluid and dynamic organization.
for instance, several proteins associate transiently with nuclear bodies under stress conditions  <cit> .
we perform analyses that help shed light on the character of compartments in terms of their proteins: known functional features, interactions and expression profiles.
moreover, we develop predictive models that are capable of predicting compartment associations of any amino acid sequence that avails itself to the nuclear environment.
we hypothesize that the high level of physical organization can be explained by advantages brought by functional modularity.
we expect that large-scale data will reflect such functional relations  as well as features that enable and implement this organization .
using the m. musculus nuclear proteome as a reference we assemble experimental data about intra-nuclear compartments.
this reference set enables us to take a system's view on a range of different properties.
firstly, we identify the compartment-specific enrichment of gene ontology terms to uncover the properties shared amongst and specific to members.
secondly, we investigate what sequence properties can be used to characterise or even identify the compartment with which a protein associates.
we build compartment-specific classifiers based on support-vector machines  to capture information in amino acid sequence data.
thirdly, we cross-reference intra-nuclear compartment and protein-protein interaction data to evaluate the extent compartments are linked via their members.
similarly, mrna levels collected over the cell cycle are used to investigate the temporal expression profile of members of each compartment.
the study focuses specifically on the dynamic nuclear bodies; cajal bodies, pml nuclear bodies, and nuclear speckles  <cit> .
we include the nucleolus that, similar to the aforementioned bodies, changes its morphology over the cell cycle.
compartments with a more static appearance, such as the nuclear pore complex, chromatin and nuclear envelope  <cit>  are, for purposes of this paper, grouped and labelled 'other'.
RESULTS
the association of proteins to intra-nuclear bodies and documented nuclear import are gleaned from multiple resources.
specifically, we use the nuclear protein database   <cit>  and generic databases including uniprotkb  <cit>  and the human protein reference database   <cit> .
we further identify proteins from the recent literature as detailed in methods.
we intersect the compartment-annotated data with the mouse nuclear proteome   <cit> .
when applicable, we map annotations for human proteins to mouse via orthology.
the final set consists of  <dig> proteins with  <dig> proteins with one or more experimentally confirmed intra-nuclear compartments .
the remaining  <dig> proteins are only known to localize generally to the nucleus.
the nucleoli is the largest compartment both physically and in terms of proteins.
of the  <dig> nuclear proteins in our reference set,  <dig> associate with the nucleoli.
<dig> associate with the nuclear speckles,  <dig> with pml nuclear bodies, and only  <dig> with cajal bodies.
these numbers reflect the variety of proteins that truly associate with each compartment, and compartment size, but also experimental biases .
it is important to note that this sampling bias may influence the results below to a smaller degree.
proteins are not exclusively associated with one compartment and this is evident in our analysis .
in fact, many proteins shuttle between them, in particular the nucleolus and nuclear speckles.
the number of different proteins belonging to a single body are shown in the diagonal.
the number of different proteins that occur in exactly two bodies are shown in the off-diagonal cells.
the row and column headers identify the bodies that share the protein.
nuclear proteins that belong to zero or two or more compartments are excluded.
analysis by annotation
in the following, we make observations of functions and biological processes of proteins that co-localize according to their annotations.
specifically, we evaluate the statistical enrichment of gene ontology  terms of proteins belonging to the same nuclear body .
go terms are expert-curated descriptors for genes and their products.
they fall into three distinct taxonomies: biological process, molecular function, and cellular component.
in table  <dig>  the statistically enriched go terms are shown at a significance level e <  <dig> .
the nucleoli are central to ribosome formation  <cit> .
much of the ribosome assembly, such as rrna transcription, occurs within the nucleoli.
in fact, previous bioinformatics analyses of proteomic data have classified nucleolar proteins into several functional groups.
approximately 30% of proteins have a function related to the production of ribosome subunits  <cit> .
the present analysis re-affirms this core activity of nucleolar proteins.
recent data have also demonstrated a range of other cellular functions of nucleoli relating to the inheritance of genetic disorders, predisposition to cancer as well as cellular senescence and stress response  <cit> .
in our set, the group of proteins that exhibit such terms within the nucleolus is not significantly over-represented.
for each compartment, go terms assigned to member proteins that are also deemed significantly enriched  are shown using the term id and the description.
the e-value is the bonferroni corrected p-value of the term as determined by the fisher's exact test .
the e-value is below  <dig>  for all terms shown.
go terms for the nuclear compartments and their parts are excluded.
other compartments have a similar functional repertoire.
indeed, and as confirmed by the go term enrichment analysis, proteins that localize to pml nuclear bodies are involved in a variety of nuclear processes including dna damage and stress response, apoptosis and cell cycle regulation  <cit> .
proteins that localize to pml nuclear bodies, partially or temporarily, are functionally diverse and the enrichment analysis identifies a few roles.
tumour suppressor proteins including tp <dig>  cbp and hipk <dig> are involved in post-translational modification processes and contribute to crucial functions of pml nuclear bodies.
for instance, phosphorylation by hipk <dig> and acetylation by cbp contribute to sensing cellular stress signals and the response to dna damage.
nuclear speckles are enriched in pre-mrna splicing factors.
in addition, nuclear speckles also house several kinases and phosphatases that phosphorylate and dephosporylate components of the splicing machinery  <cit> .
the overall role of splicing speckles aligns with the regulation of splicing factors' access to the splicing machinery .
the go term with the strongest enrichment for cajal bodies is 'spliceosome assembly' at e <  <dig> .
the weak enrichment is probably caused by the small and incomplete set of proteins known to localize to these bodies.
we thus make a few anecdotal remarks.
there is growing evidence implicating cajal bodies with snrnp and snornp biogenesis  <cit> .
the presence of tp <dig> in cajal bodies during stress suggests the potential involvement of this compartment in the cellular response to stress  <cit> .
that cajal bodies contain several nucleolar proteins  suggest that they are involved in preribosomal rna processing  <cit> .
additionally, cajal bodies are implicated in histone pre-mrna 3' maturation and basal transcription factor of rna polymerases i, ii and iii and telomerase rna  <cit> .
analysis by sequence
this section explores pre-defined sequence patterns relating to intra-nuclear organization, potentially specific to compartments, namely nuclear localization signals and sumoylation sites.
this section also investigates more subtle sequence features as compartment determinants by training classifiers that are evaluated  and then used to characterize proteins with no presently known nuclear compartment.
nuclear localization signals  are short amino acid sequences that induce nuclear import by binding to so-called importins.
nls-based import is believed to be the most common  mechanism for proteins to enter the nucleus but not the only one.
to explore the effect of nls on intra-nuclear sorting, we identify nls-carrying nuclear proteins by matching the amino acid sequence to the  <dig> motifs in nlsdb  <cit> .
bickmore and surtherland  <cit>  report that by using psort they detect nls in 80-86% proteins that localize to nucleoli, nuclear speckles and pml nuclear bodies; and 62% of proteins that associate with cajal bodies.
nlsdb is more strict and a smaller number of positive predictions are expected generally.
only nuclear speckles show a larger proportion of nlss than by chance alone .
at 38%, nls-containing proteins are more common in speckles than in other nuclear bodies.
other bodies have 29-34% nls-containing proteins.
sumoylation is a post-translational modification of proteins, attaching a small ubiquitin-like modifier protein  to a lysine in the substrate.
evidence suggests that sumoylation plays a role in preserving function and integrity of intra-nuclear compartments  <cit> .
for instance, many of the proteins localize to pml nuclear bodies are reported to be sumoylated  <cit> , and sumoylation has been suggested to be responsible for the assembly of these bodies  <cit> .
for instance, sumoylation is necessary for the recruitment of pml nuclear bodies components such as sp <dig>
to explore the role of sumoylation in the organization of compartments, proteins that contain a putative sumoylation site are identified.
the sumoylation consensus motif is defined as Ψ-k-x-e .
this motif is used to scan all proteins, and all proteins with at least one hit are considered positives.
overall, 41% of nuclear proteins have this motif.
we employ a statistical test to observe which bodies that are significantly enriched for the sumoylation motif .
the analysis shows that sumoylation predominantly occurs in pml nuclear bodies .
the other compartments are not significantly enriched with sumoylated proteins .
the existence of compartment-specific sequence features is further evaluated using svm machine learning classifiers.
svms have been successfully applied to discover and leverage sequence similarities not easily represented as linear motifs to build predictive models.
to investigate if there are compartment-specific determinants in the amino acid sequences, we build classifiers from the available data and evaluate their ability to generalise to unseen proteins.
classifiers with high accuracy can be used to understand yet unclassified sequences, e.g.
nuclear proteins with unknown intra-nuclear associations.
the development of the prediction methods for intra-nuclear proteins is exemplified by work by lei and dai  <cit> .
they predict a single intra-nuclear localization using svms.
multi-localized proteins are excluded and thus their model ignores the large and important fraction of proteins that transiently associate with several compartments.
lei and dai report an accuracy as measured by the correlation between the prediction and the actual compartment ranging from  <dig>  to  <dig>  .
we include and handle multi-localized proteins.
specifically, we cast compartment prediction as multiple binary classification problems, where proteins of one nuclear body are labeled as positives and all proteins from other compartments are negatives .
different configurations are tested and we report only on the best below .
we set aside the set of proteins that have no intra-nuclear location assigned.
to illustrate the overall performance of classification on basis of sequence data, we generate receiver operating characteristic  curves for the svms equipped with the local alignment kernel  <cit> .
the roc curve illustrates the rate of true positives as a function of the rate of false positives .
in figure  <dig>  we note that the classification accuracy is high for nucleoli and nuclear speckles.
slightly lower performance is achieved for the functionally heterogeneous pml nuclear bodies.
cajal bodies proteins are classified poorly.
the positive sets are very small for the latter two nuclear bodies , a property that is likely to adversely affect classification accuracy.
we note the area under the roc curve  in table  <dig> determined for held-out data  for each body.
an auc of  <dig>  for nuclear speckles and  <dig>  for nucleolar proteins indicate relatively high accuracy  and that distinguishing sequence features exist.
the mean accuracies and standard deviations for compartment-specific classifiers when tested on data with-held during training.
all classifiers use the local alignment kernel and tests are repeated ten times with different data set divisions.
each classifier is next used to screen the  <dig> protein set with no known intra-nuclear location.
to provide a reasonable estimate of total numbers of compartment members, we first determine a suitable threshold.
specifically, we set the svm's threshold such that approximately the same number of positives would have been generated on the data for which intra-nuclear compartments are known.
the thresholding of the un-annotated novel set results in  <dig> nucleoli proteins,  <dig> nuclear speckles proteins and  <dig> pml nuclear bodies proteins and  <dig> cajal bodies proteins of the  <dig> un-annotated protein set .
gleaning the annotated data, we calculate the expected false discovery rate  for each compartment.
for nucleoli and nuclear speckles, the fdr is  <dig>  and  <dig> , respectively.
hence, of the  <dig> or so predicted positives, about 40% are expected to be incorrect.
in contrast, the pml nuclear bodies and cajal bodies predictors have an fdr of  <dig>  and  <dig> , respectively.
it is important to note that the negative set is much larger than the positive set for each of these compartments, naturally leading to many false discoveries.
we also expect there to be a large number of proteins in the training set that due to the lack of experimental data are wrongly labelled as negatives.
sequence features that are found by predictors potentially correspond to functional properties of proteins.
we are therefore interested in seeing to what extent such features are shared between proteins of different compartments.
from the accuracy of prediction we already know that nucleolar and nuclear speckle proteins are the easiest to predict.
more detail is found in table  <dig> that shows the number of proteins that are predicted by each compartment-specific predictor.
specifically, by using the same thresholds as we set above, we determine for each predictor, how many predicted proteins that are in fact false positives, and belong to another compartment.
it appears that a high proportion of cajal body members are positively identified by the nucleolus predictor  indicating that nucleolar proteins may share features with the small set of cajal body proteins.
indeed, some cajal body components including fibrillarin are located in cajal body before accumulating in nucleoli  <cit> .
in neurons, approximately 30% of cajal body proteins are attached to the nucleolus, suggesting that the association may play an important role in delivering snornps to the nucleolus  <cit> .
similarly, 24/ <dig> = 29% of all pml body proteins are predicted by the nucleolus predictor.
we also note that the pml body predictor identifies 22/ <dig> = 6% of all nuclear speckle proteins, and that the nuclear speckle predictor finds 20% of all pml body proteins and 20% of all cajal body proteins.
it is interesting to note that pml bodies are often physically juxtaposed with both nuclear speckles and cajal bodies  <cit> .
short actin- or lamin-based fibres have been suggested to transiently link such domains  <cit> .
overall, we are unable to fully explain the occurrence of false positives, but there appears to be biologically meaningful circumstances for the confusion.
for each compartment-specific predictor, the known  compartment is shown for all "positive" predictions .
the last column shows the number of predictions that knowingly belong to the compartment identified in the second column.
for example, the test outcomes of the predictor trained to discriminate nucleolar proteins from others are shown in the first section.
of  <dig> positives,  <dig> were correctly classified,  <dig> were classified as "nuclear speckle" proteins, etc.
note that in the case of multi-class proteins, the alternative compartment is not necessarily incorrect.
analysis by protein-protein interaction data
it is widely held that proteins associate with intra-nuclear compartments via molecular interaction.
however, interaction is often transient and proteins move between compartments by anomalous diffusion  <cit> .
we are interested in understanding if there is a pattern of "interaction exchange" between compartments reflected by current data collections.
this section inspects the interactions of individual proteins, given their association with intra-nuclear compartments.
in particular, we ask, to what extent is there pairwise interactions between members of different compartments?
we consider the extensive protein-protein interaction data from hprd  <cit>  and map this data to mouse proteins by orthology .
to simplify the analysis, we include only interactions between proteins that are each annotated with a single nuclear body or compartment.
this means that each interaction is strictly either intra- or inter-compartmental.
the number of intra-compartmental interactions are shown in the diagonal.
the number of inter-compartmental interactions are shown in the off-diagonal cells .
intra-compartmental interaction is typically far more frequent than any single inter-compartmental interaction.
this general observation supports that protein-protein interaction is a primary force behind intra-nuclear organization.
due to the small number of samples, specific observations involving two different locations are not supported statistically.
our analysis only concerns nuclear imported proteins, but we note that the protein interaction data can contain sampling biases  <cit>  at a fine-grained, "compartment" level.
we inspected the supporting literature for a subset  of the interaction entries and in no case did the experiment design explicitly involve intra-nuclear compartments.
we believe that the risk is low that interaction profiles for compartments are artificially skewed.
analysis by cell cycle expression
the cell cycle is a major and critical biological process in the cell, representing the stages of replication  to cell division .
understanding how intra-nuclear bodies relate to the stages uncovers possible links to other cell cycle components and elucidates the compartments' regulatory roles.
during the cell cycle, most intra-nuclear bodies change profoundly in terms of their biochemical composition.
assembly and disassembly of some compartments appear to correlate with cell cycle stage .
some genes are only expressed at a specific stage of the cell cycle, allowing us to investigate if proteins for a compartment tend to avail themselves selectively during the cell cycle.
specifically, we look closer at the proportion of proteins associated with each compartment that are periodically expressed in the cell cycle.
cyclebase contains the expression of human genes as measured during the cell cycle  <cit> .
it provides for each gene an assessment whether it is differentially expressed and the periodicity of such expression .
first, human genes are mapped to their mouse orthologs.
we then extracted the peaktime of expression for each gene, incorporating interphase  and mitosis .
in total,  <dig> genes  have a peaktime assigned.
out of this set,  <dig> and  <dig> genes code for nuclear speckle and nucleolar proteins, respectively.
for pml nuclear bodies and cajal bodies, we find only  <dig> and  <dig> genes with a peaktime assigned, respectively.
the analysis below must thus be interpreted in light of these limited numbers.
nucleoli
nucleoli are dynamic structures that assemble in interphase during transcription.
as seen in figure  <dig>  nucleoli-associated periodic genes are indeed expressed in interphase with a slightly bi-modal distribution .
as an example, cdk <dig> and cdk <dig>  are expressed at this time, modifying and thereby controlling other proteins.
cdk <dig> is also expressed during mitosis.
nucleoli also contain proteins controlling dna replication.
some of the responsible genes are selectively expressed in s phase are members of the minichromosome maintenance  protein family .
mcm <dig> is also observed to express in m phase suggesting a role beyond replication.
nucleolus stress response is exemplified by the periodic expression of cdkn2a in s phase.
cdkn2a interacts with and sequesters mdm <dig> and p19arf proteins to indirectly activate tumor suppressor protein tp <dig>  <cit> .
nuclear speckles
nuclear speckles recruit many transcription and splicing factors.
only  <dig> of  <dig> nuclear speckle proteins have genes that are periodically expressed.
57% and 34% of these  <dig> genes are expressed in g <dig> and s phase, respectively.
<dig> genes are expressed in g <dig> and  <dig> in m phase .
any clear pattern is therefore not seen in figure  <dig>  anecdotally, we note that at m/g <dig>  nxf <dig> and hspa <dig> are expressed.
nxf <dig> is a gene that is involven in the export of mrna from nucleolus to cytoplasm.
hspa <dig> is a member of the heat shock protein family and functions as a molecular chaperone for newly synthesized proteins  <cit> .
in g <dig> phase, several genes that encode proteins with diverse functions are expressed, including rbm <dig>  syncrip and zranb <dig>  rbm <dig> is implicated as a nuclear receptor coactivator, enhancing the transcription process through other coactivators.
syncrip is important for mrna processing, translation coupled mrna turnover and splicing  <cit> .
zranb <dig> is a zinc finger protein that is required for alternative splicing.
this protein exists as at least two splice variants and may interfere with constitutive 5'-splice site selection  <cit> .
pml nuclear bodies
pml nuclear bodies are believed to mediate dna transcription and dna damage response  <cit> .
during the cell cycle,  <dig> of  <dig> pml body genes with peaktime assigned are expressed in s phase.
among those genes, several genes that play an essential role in transcription are expressed in the early stage of this phase, including runx <dig> and zfp <dig>  the precise mechanism of how pml nuclear bodies control transcription is unclear.
zhong et al.
report that pml nuclear bodies modulate the availability of transcription factors  <cit> .
another study suggests that pml nuclear bodies are involved in chromatin remodelling processes  <cit> .
changes to composition and location of pml nuclear bodies during the cell cycle appear to influence transcription  <cit> .
cell cycle checkpoint proteins verify that processes at each phase are accurately completed before progressing to the next.
in our data we find topbp <dig> and chek <dig> that respond to cellular stress .
proteins encoded by these genes are also putative tumor suppressors.
these proteins have a forkhead-associated protein interaction domain that is important for activation in response to dna damage.
in s phase, these proteins also co-localize with brca <dig> to restore survival after dna damage.
topbp <dig> is also involved in initiation of dna replication.
several multi-compartment proteins are coded by genes periodically expressed during the cell cycle.
nono, cdk <dig> and dhx <dig> localize to nucleoli and nuclear speckles.
topbp <dig> and cbx <dig> localize to both nucleoli and pml nuclear bodies.
nono is dna and rna binding protein, and enhances the binding of conventional sequence-specific transcription factors to their recognition sites  <cit> .
discussion
the availability of large-scale data of intra-nuclear proteins provides a unique opportunity to explore different features for characterizing individual compartments on basis of their protein complement and for discriminating proteins of one compartment from another.
exploration of several features including gene annotations, amino acid sequence, protein-protein interaction data, and cell cycle expression sheds light on the functional organization of physically embodied intra-nuclear compartments.
here, we discuss our "data-driven" analyses compartment by compartment, after a few general observations.
using gene ontology term enrichment analysis, the functional homogeneity of a group of gene products can be explored.
for each compartment, we collect the constituent proteins that are identified by recent experimental work, and determine significant go terms in a nuclear protein context.
similarly, we investigate the enrichment of two sequence features, essential for nuclear import  and nuclear organization .
our analyses also illustrate the relationships between compartments.
the fluid exchange of many proteins illustrate a functional relationship.
we note that almost all bodies share proteins.
additionally, interactions between proteins  are found both within compartments and between compartments.
the large proportion of observed intra-compartment interactions support that protein-protein interaction is a major driver for intra-nuclear organization.
amino acid composition and subtle sequence similarities between proteins of different compartments are also potential indicators of functional similarity.
we build classifiers that are able to detect such features.
their inability to distinguish between two compartments illustrates the overlap and sharing of sequence features of proteins associated with those two compartments .
in particular, we note that the nucleolar predictor picks up an inproportionate number of pml body and cajal body proteins .
it also seems the pml body predictor is confounded by nuclear speckle proteins and vice versa.
we note that several studies suggest that these bodies are indeed physically juxtaposed or transiently linked in some situations  <cit> .
nucleolus
broadly, we confirm previous observations that the nucleolus is heavily involved in ribosomal biogenesis.
there are  <dig> known nucleolar proteins in our training set, but the nucleolus classifier predicts that an additional 50%,  <dig> proteins are missing.
differential expression of nucleolar genes is seen in s and g <dig> stages of the cell cycle.
anecdotal observations of periodically expressed genes identify roles for the nucleolus in stress response and dna replication.
nuclear speckles
in line with previous studies, annotation enrichment analysis of nuclear speckle proteins identify rna splicing as the core activity of this compartment.
it is also significantly enriched in nls-carrying proteins  relative other nuclear-imported proteins, indicating a relationship with the classical nuclear import machinery .
using our nuclear speckle classifier, we suggest that there are many more nuclear speckle proteins to be discovered experimentally.
currently,  <dig> are known but another  <dig>  are predicted.
periodic expression was only found for 21/ <dig> genes, primarily expressed during the g <dig> and s phases.
pml nuclear body
annotations of pml nuclear body proteins show an enrichment of terms relating to cell cycle control, stress response and dna repair--all in agreement with the literature.
the term "protein modification process" is also identified, lending support to the hypothesis that sumoylation is critical for this nuclear body  <cit> .
the prevalence of sumoylation motifs in member proteins is also higher than in any of the other compartments.
on top of the  <dig> known, about  <dig> new pml nuclear proteins are predicted but the classification accuracy is poor.
<dig> of the  <dig> genes responsible for the pml body proteins are periodically expressed, mostly during the s phase.
cajal body
only  <dig> proteins are known to associate with cajal bodies.
we find no significant go terms at e <  <dig> , but "spliceosome assembly" at e <  <dig>  with such a limited data set, building a classifier is premature.
we estimate, however, that another  <dig> cajal body proteins are still to be discovered .
CONCLUSIONS
we expected that the data-driven analyses would demonstrate that intra-nuclear compartments are functionally homogeneous and that their members would exhibit properties that enable their sorting.
the collected large-scale protein data provides only partial evidence that the physical organization of the nucleus mirrors functional modularity.
the current data set is far from completely annotated, suggesting that our methods for evaluating prediction accuracy are not robust.
the error rates may be lower than what we have been able to conclude-ultimately we should instead rely on experimental validation.
with the incomplete "trusted" data set, we fail to unambiguously identify proteins' intra-nuclear destination, suggesting that critical drivers behind of intra-nuclear translocation are yet to be identified.

 the nucleus not only houses the genetic material but also administers its transcription. morphologically defined intra-nuclear compartments or bodies are non-randomly interspersed amongst chromosomes. preliminary evidence indicates that these compartments are spatially and functionally organized, and play important regulatory roles. for example, nucleoli are primarily concerned with ribosomal biogenesis at sites of ribosomal genes, nuclear speckles accumulate pre-mrna splicing factors at actively transcribed genes, and nuclear pore complexes sit in the nuclear membrane to control import and export of material. other compartments, such as cajal and pml nuclear bodies, appear to be involved in a much broader range of functions. the modular nature of the nuclear milieu and the fluid means of molecular transport enable several levels of regulation. for example, pml nuclear bodies assemble in response to sumoylation. they also retain and release regulatory proteins in response to cellular stress. similarly, the nucleolus sequesters regulatory proteins for timely release. at a more general level, nuclear pores regulate protein access to the nucleus. this paper paves the way for the computational mapping of the role of nuclear architecture in critical regulatory processes. it taps recent experimental data and the growing knowledge of nuclear architecture to portray the function and the protein complement of intra-nuclear compartments. it is important to note that proteins and rna do not constitutively and exclusively localize to a single nuclear compartment. data sets and models need to account for this biochemically fluid and dynamic organization. for instance, several proteins associate transiently with nuclear bodies under stress conditions. we perform analyses that help shed light on the character of compartments in terms of their proteins: known functional features, interactions and expression profiles. moreover, we develop predictive models that are capable of predicting compartment associations of any amino acid sequence that avails itself to the nuclear environment. we hypothesize that the high level of physical organization can be explained by advantages brought by functional modularity. we expect that large-scale data will reflect such functional relations as well as features that enable and implement this organization. using the m. musculus nuclear proteome as a reference we assemble experimental data about intra-nuclear compartments. this reference set enables us to take a system's view on a range of different properties. firstly, we identify the compartment-specific enrichment of gene ontology terms to uncover the properties shared amongst and specific to members. secondly, we investigate what sequence properties can be used to characterise or even identify the compartment with which a protein associates. we build compartment-specific classifiers based on support-vector machines to capture information in amino acid sequence data. thirdly, we cross-reference intra-nuclear compartment and protein-protein interaction data to evaluate the extent compartments are linked via their members. similarly, mrna levels collected over the cell cycle are used to investigate the temporal expression profile of members of each compartment. the study focuses specifically on the dynamic nuclear bodies; cajal bodies, pml nuclear bodies, and nuclear speckles. we include the nucleolus that, similar to the aforementioned bodies, changes its morphology over the cell cycle. compartments with a more static appearance, such as the nuclear pore complex, chromatin and nuclear envelope are, for purposes of this paper, grouped and labelled'other'.  the association of proteins to intra-nuclear bodies and documented nuclear import are gleaned from multiple resources. specifically, we use the nuclear protein database and generic databases including uniprotkb and the human protein reference database. we further identify proteins from the recent literature as detailed in methods. we intersect the compartment-annotated data with the mouse nuclear proteome. when applicable, we map annotations for human proteins to mouse via orthology. the final set consists of proteins with proteins with one or more experimentally confirmed intra-nuclear compartments. the remaining proteins are only known to localize generally to the nucleus. the nucleoli is the largest compartment both physically and in terms of proteins. of the nuclear proteins in our reference set, associate with the nucleoli. associate with the nuclear speckles, with pml nuclear bodies, and only with cajal bodies. these numbers reflect the variety of proteins that truly associate with each compartment, and compartment size, but also experimental biases. it is important to note that this sampling bias may influence the results below to a smaller degree. proteins are not exclusively associated with one compartment and this is evident in our analysis. in fact, many proteins shuttle between them, in particular the nucleolus and nuclear speckles. the number of different proteins belonging to a single body are shown in the diagonal. the number of different proteins that occur in exactly two bodies are shown in the off-diagonal cells. the row and column headers identify the bodies that share the protein. nuclear proteins that belong to zero or two or more compartments are excluded. analysis by annotation in the following, we make observations of functions and biological processes of proteins that co-localize according to their annotations. specifically, we evaluate the statistical enrichment of gene ontology terms of proteins belonging to the same nuclear body. go terms are expert-curated descriptors for genes and their products. they fall into three distinct taxonomies: biological process, molecular function, and cellular component. in table the statistically enriched go terms are shown at a significance level e <. the nucleoli are central to ribosome formation. much of the ribosome assembly, such as rrna transcription, occurs within the nucleoli. in fact, previous bioinformatics analyses of proteomic data have classified nucleolar proteins into several functional groups. approximately 30% of proteins have a function related to the production of ribosome subunits. the present analysis re-affirms this core activity of nucleolar proteins. recent data have also demonstrated a range of other cellular functions of nucleoli relating to the inheritance of genetic disorders, predisposition to cancer as well as cellular senescence and stress response. in our set, the group of proteins that exhibit such terms within the nucleolus is not significantly over-represented. for each compartment, go terms assigned to member proteins that are also deemed significantly enriched are shown using the term id and the description. the e-value is the bonferroni corrected p-value of the term as determined by the fisher's exact test. the e-value is below for all terms shown. go terms for the nuclear compartments and their parts are excluded. other compartments have a similar functional repertoire. indeed, and as confirmed by the go term enrichment analysis, proteins that localize to pml nuclear bodies are involved in a variety of nuclear processes including dna damage and stress response, apoptosis and cell cycle regulation. proteins that localize to pml nuclear bodies, partially or temporarily, are functionally diverse and the enrichment analysis identifies a few roles. tumour suppressor proteins including tp cbp and hipk are involved in post-translational modification processes and contribute to crucial functions of pml nuclear bodies. for instance, phosphorylation by hipk and acetylation by cbp contribute to sensing cellular stress signals and the response to dna damage. nuclear speckles are enriched in pre-mrna splicing factors. in addition, nuclear speckles also house several kinases and phosphatases that phosphorylate and dephosporylate components of the splicing machinery. the overall role of splicing speckles aligns with the regulation of splicing factors' access to the splicing machinery. the go term with the strongest enrichment for cajal bodies is'spliceosome assembly' at e <. the weak enrichment is probably caused by the small and incomplete set of proteins known to localize to these bodies. we thus make a few anecdotal remarks. there is growing evidence implicating cajal bodies with snrnp and snornp biogenesis. the presence of tp in cajal bodies during stress suggests the potential involvement of this compartment in the cellular response to stress. that cajal bodies contain several nucleolar proteins suggest that they are involved in preribosomal rna processing. additionally, cajal bodies are implicated in histone pre-mrna 3' maturation and basal transcription factor of rna polymerases i, ii and iii and telomerase rna. analysis by sequence this section explores pre-defined sequence patterns relating to intra-nuclear organization, potentially specific to compartments, namely nuclear localization signals and sumoylation sites. this section also investigates more subtle sequence features as compartment determinants by training classifiers that are evaluated and then used to characterize proteins with no presently known nuclear compartment. nuclear localization signals are short amino acid sequences that induce nuclear import by binding to so-called importins. nls-based import is believed to be the most common mechanism for proteins to enter the nucleus but not the only one. to explore the effect of nls on intra-nuclear sorting, we identify nls-carrying nuclear proteins by matching the amino acid sequence to the motifs in nlsdb. bickmore and surtherland report that by using psort they detect nls in 80-86% proteins that localize to nucleoli, nuclear speckles and pml nuclear bodies; and 62% of proteins that associate with cajal bodies. nlsdb is more strict and a smaller number of positive predictions are expected generally. only nuclear speckles show a larger proportion of nlss than by chance alone. at 38%, nls-containing proteins are more common in speckles than in other nuclear bodies. other bodies have 29-34% nls-containing proteins. sumoylation is a post-translational modification of proteins, attaching a small ubiquitin-like modifier protein to a lysine in the substrate. evidence suggests that sumoylation plays a role in preserving function and integrity of intra-nuclear compartments. for instance, many of the proteins localize to pml nuclear bodies are reported to be sumoylated, and sumoylation has been suggested to be responsible for the assembly of these bodies. for instance, sumoylation is necessary for the recruitment of pml nuclear bodies components such as sp to explore the role of sumoylation in the organization of compartments, proteins that contain a putative sumoylation site are identified. the sumoylation consensus motif is defined as Ψ-k-x-e. this motif is used to scan all proteins, and all proteins with at least one hit are considered positives. overall, 41% of nuclear proteins have this motif. we employ a statistical test to observe which bodies that are significantly enriched for the sumoylation motif. the analysis shows that sumoylation predominantly occurs in pml nuclear bodies. the other compartments are not significantly enriched with sumoylated proteins. the existence of compartment-specific sequence features is further evaluated using svm machine learning classifiers. svms have been successfully applied to discover and leverage sequence similarities not easily represented as linear motifs to build predictive models. to investigate if there are compartment-specific determinants in the amino acid sequences, we build classifiers from the available data and evaluate their ability to generalise to unseen proteins. classifiers with high accuracy can be used to understand yet unclassified sequences, e.g. nuclear proteins with unknown intra-nuclear associations. the development of the prediction methods for intra-nuclear proteins is exemplified by work by lei and dai. they predict a single intra-nuclear localization using svms. multi-localized proteins are excluded and thus their model ignores the large and important fraction of proteins that transiently associate with several compartments. lei and dai report an accuracy as measured by the correlation between the prediction and the actual compartment ranging from to. we include and handle multi-localized proteins. specifically, we cast compartment prediction as multiple binary classification problems, where proteins of one nuclear body are labeled as positives and all proteins from other compartments are negatives. different configurations are tested and we report only on the best below. we set aside the set of proteins that have no intra-nuclear location assigned. to illustrate the overall performance of classification on basis of sequence data, we generate receiver operating characteristic curves for the svms equipped with the local alignment kernel. the roc curve illustrates the rate of true positives as a function of the rate of false positives. in figure we note that the classification accuracy is high for nucleoli and nuclear speckles. slightly lower performance is achieved for the functionally heterogeneous pml nuclear bodies. cajal bodies proteins are classified poorly. the positive sets are very small for the latter two nuclear bodies, a property that is likely to adversely affect classification accuracy. we note the area under the roc curve in table determined for held-out data for each body. an auc of for nuclear speckles and for nucleolar proteins indicate relatively high accuracy and that distinguishing sequence features exist. the mean accuracies and standard deviations for compartment-specific classifiers when tested on data with-held during training. all classifiers use the local alignment kernel and tests are repeated ten times with different data set divisions. each classifier is next used to screen the protein set with no known intra-nuclear location. to provide a reasonable estimate of total numbers of compartment members, we first determine a suitable threshold. specifically, we set the svm's threshold such that approximately the same number of positives would have been generated on the data for which intra-nuclear compartments are known. the thresholding of the un-annotated novel set results in nucleoli proteins, nuclear speckles proteins and pml nuclear bodies proteins and cajal bodies proteins of the un-annotated protein set. gleaning the annotated data, we calculate the expected false discovery rate for each compartment. for nucleoli and nuclear speckles, the fdr is and, respectively. hence, of the or so predicted positives, about 40% are expected to be incorrect. in contrast, the pml nuclear bodies and cajal bodies predictors have an fdr of and, respectively. it is important to note that the negative set is much larger than the positive set for each of these compartments, naturally leading to many false discoveries. we also expect there to be a large number of proteins in the training set that due to the lack of experimental data are wrongly labelled as negatives. sequence features that are found by predictors potentially correspond to functional properties of proteins. we are therefore interested in seeing to what extent such features are shared between proteins of different compartments. from the accuracy of prediction we already know that nucleolar and nuclear speckle proteins are the easiest to predict. more detail is found in table that shows the number of proteins that are predicted by each compartment-specific predictor. specifically, by using the same thresholds as we set above, we determine for each predictor, how many predicted proteins that are in fact false positives, and belong to another compartment. it appears that a high proportion of cajal body members are positively identified by the nucleolus predictor indicating that nucleolar proteins may share features with the small set of cajal body proteins. indeed, some cajal body components including fibrillarin are located in cajal body before accumulating in nucleoli. in neurons, approximately 30% of cajal body proteins are attached to the nucleolus, suggesting that the association may play an important role in delivering snornps to the nucleolus. similarly, 24/ = 29% of all pml body proteins are predicted by the nucleolus predictor. we also note that the pml body predictor identifies 22/ = 6% of all nuclear speckle proteins, and that the nuclear speckle predictor finds 20% of all pml body proteins and 20% of all cajal body proteins. it is interesting to note that pml bodies are often physically juxtaposed with both nuclear speckles and cajal bodies. short actin- or lamin-based fibres have been suggested to transiently link such domains. overall, we are unable to fully explain the occurrence of false positives, but there appears to be biologically meaningful circumstances for the confusion. for each compartment-specific predictor, the known compartment is shown for all "positive" predictions. the last column shows the number of predictions that knowingly belong to the compartment identified in the second column. for example, the test outcomes of the predictor trained to discriminate nucleolar proteins from others are shown in the first section. of positives, were correctly classified, were classified as "nuclear speckle" proteins, etc. note that in the case of multi-class proteins, the alternative compartment is not necessarily incorrect. analysis by protein-protein interaction data it is widely held that proteins associate with intra-nuclear compartments via molecular interaction. however, interaction is often transient and proteins move between compartments by anomalous diffusion. we are interested in understanding if there is a pattern of "interaction exchange" between compartments reflected by current data collections. this section inspects the interactions of individual proteins, given their association with intra-nuclear compartments. in particular, we ask, to what extent is there pairwise interactions between members of different compartments? we consider the extensive protein-protein interaction data from hprd and map this data to mouse proteins by orthology. to simplify the analysis, we include only interactions between proteins that are each annotated with a single nuclear body or compartment. this means that each interaction is strictly either intra- or inter-compartmental. the number of intra-compartmental interactions are shown in the diagonal. the number of inter-compartmental interactions are shown in the off-diagonal cells. intra-compartmental interaction is typically far more frequent than any single inter-compartmental interaction. this general observation supports that protein-protein interaction is a primary force behind intra-nuclear organization. due to the small number of samples, specific observations involving two different locations are not supported statistically. our analysis only concerns nuclear imported proteins, but we note that the protein interaction data can contain sampling biases at a fine-grained, "compartment" level. we inspected the supporting literature for a subset of the interaction entries and in no case did the experiment design explicitly involve intra-nuclear compartments. we believe that the risk is low that interaction profiles for compartments are artificially skewed. analysis by cell cycle expression the cell cycle is a major and critical biological process in the cell, representing the stages of replication to cell division. understanding how intra-nuclear bodies relate to the stages uncovers possible links to other cell cycle components and elucidates the compartments' regulatory roles. during the cell cycle, most intra-nuclear bodies change profoundly in terms of their biochemical composition. assembly and disassembly of some compartments appear to correlate with cell cycle stage. some genes are only expressed at a specific stage of the cell cycle, allowing us to investigate if proteins for a compartment tend to avail themselves selectively during the cell cycle. specifically, we look closer at the proportion of proteins associated with each compartment that are periodically expressed in the cell cycle. cyclebase contains the expression of human genes as measured during the cell cycle. it provides for each gene an assessment whether it is differentially expressed and the periodicity of such expression. first, human genes are mapped to their mouse orthologs. we then extracted the peaktime of expression for each gene, incorporating interphase and mitosis. in total, genes have a peaktime assigned. out of this set, and genes code for nuclear speckle and nucleolar proteins, respectively. for pml nuclear bodies and cajal bodies, we find only and genes with a peaktime assigned, respectively. the analysis below must thus be interpreted in light of these limited numbers. nucleoli nucleoli are dynamic structures that assemble in interphase during transcription. as seen in figure nucleoli-associated periodic genes are indeed expressed in interphase with a slightly bi-modal distribution. as an example, cdk and cdk are expressed at this time, modifying and thereby controlling other proteins. cdk is also expressed during mitosis. nucleoli also contain proteins controlling dna replication. some of the responsible genes are selectively expressed in s phase are members of the minichromosome maintenance protein family. mcm is also observed to express in m phase suggesting a role beyond replication. nucleolus stress response is exemplified by the periodic expression of cdkn2a in s phase. cdkn2a interacts with and sequesters mdm and p19arf proteins to indirectly activate tumor suppressor protein tp. nuclear speckles nuclear speckles recruit many transcription and splicing factors. only of nuclear speckle proteins have genes that are periodically expressed. 57% and 34% of these genes are expressed in g and s phase, respectively. genes are expressed in g and in m phase. any clear pattern is therefore not seen in figure anecdotally, we note that at m/g nxf and hspa are expressed. nxf is a gene that is involven in the export of mrna from nucleolus to cytoplasm. hspa is a member of the heat shock protein family and functions as a molecular chaperone for newly synthesized proteins. in g phase, several genes that encode proteins with diverse functions are expressed, including rbm syncrip and zranb rbm is implicated as a nuclear receptor coactivator, enhancing the transcription process through other coactivators. syncrip is important for mrna processing, translation coupled mrna turnover and splicing. zranb is a zinc finger protein that is required for alternative splicing. this protein exists as at least two splice variants and may interfere with constitutive 5'-splice site selection. pml nuclear bodies pml nuclear bodies are believed to mediate dna transcription and dna damage response. during the cell cycle, of pml body genes with peaktime assigned are expressed in s phase. among those genes, several genes that play an essential role in transcription are expressed in the early stage of this phase, including runx and zfp the precise mechanism of how pml nuclear bodies control transcription is unclear. zhong et al. report that pml nuclear bodies modulate the availability of transcription factors. another study suggests that pml nuclear bodies are involved in chromatin remodelling processes. changes to composition and location of pml nuclear bodies during the cell cycle appear to influence transcription. cell cycle checkpoint proteins verify that processes at each phase are accurately completed before progressing to the next. in our data we find topbp and chek that respond to cellular stress. proteins encoded by these genes are also putative tumor suppressors. these proteins have a forkhead-associated protein interaction domain that is important for activation in response to dna damage. in s phase, these proteins also co-localize with brca to restore survival after dna damage. topbp is also involved in initiation of dna replication. several multi-compartment proteins are coded by genes periodically expressed during the cell cycle. nono, cdk and dhx localize to nucleoli and nuclear speckles. topbp and cbx localize to both nucleoli and pml nuclear bodies. nono is dna and rna binding protein, and enhances the binding of conventional sequence-specific transcription factors to their recognition sites. discussion the availability of large-scale data of intra-nuclear proteins provides a unique opportunity to explore different features for characterizing individual compartments on basis of their protein complement and for discriminating proteins of one compartment from another. exploration of several features including gene annotations, amino acid sequence, protein-protein interaction data, and cell cycle expression sheds light on the functional organization of physically embodied intra-nuclear compartments. here, we discuss our "data-driven" analyses compartment by compartment, after a few general observations. using gene ontology term enrichment analysis, the functional homogeneity of a group of gene products can be explored. for each compartment, we collect the constituent proteins that are identified by recent experimental work, and determine significant go terms in a nuclear protein context. similarly, we investigate the enrichment of two sequence features, essential for nuclear import and nuclear organization. our analyses also illustrate the relationships between compartments. the fluid exchange of many proteins illustrate a functional relationship. we note that almost all bodies share proteins. additionally, interactions between proteins are found both within compartments and between compartments. the large proportion of observed intra-compartment interactions support that protein-protein interaction is a major driver for intra-nuclear organization. amino acid composition and subtle sequence similarities between proteins of different compartments are also potential indicators of functional similarity. we build classifiers that are able to detect such features. their inability to distinguish between two compartments illustrates the overlap and sharing of sequence features of proteins associated with those two compartments. in particular, we note that the nucleolar predictor picks up an inproportionate number of pml body and cajal body proteins. it also seems the pml body predictor is confounded by nuclear speckle proteins and vice versa. we note that several studies suggest that these bodies are indeed physically juxtaposed or transiently linked in some situations. nucleolus broadly, we confirm previous observations that the nucleolus is heavily involved in ribosomal biogenesis. there are known nucleolar proteins in our training set, but the nucleolus classifier predicts that an additional 50%, proteins are missing. differential expression of nucleolar genes is seen in s and g stages of the cell cycle. anecdotal observations of periodically expressed genes identify roles for the nucleolus in stress response and dna replication. nuclear speckles in line with previous studies, annotation enrichment analysis of nuclear speckle proteins identify rna splicing as the core activity of this compartment. it is also significantly enriched in nls-carrying proteins relative other nuclear-imported proteins, indicating a relationship with the classical nuclear import machinery. using our nuclear speckle classifier, we suggest that there are many more nuclear speckle proteins to be discovered experimentally. currently, are known but another are predicted. periodic expression was only found for 21/ genes, primarily expressed during the g and s phases. pml nuclear body annotations of pml nuclear body proteins show an enrichment of terms relating to cell cycle control, stress response and dna repair--all in agreement with the literature. the term "protein modification process" is also identified, lending support to the hypothesis that sumoylation is critical for this nuclear body. the prevalence of sumoylation motifs in member proteins is also higher than in any of the other compartments. on top of the known, about new pml nuclear proteins are predicted but the classification accuracy is poor. of the genes responsible for the pml body proteins are periodically expressed, mostly during the s phase. cajal body only proteins are known to associate with cajal bodies. we find no significant go terms at e <, but "spliceosome assembly" at e < with such a limited data set, building a classifier is premature. we estimate, however, that another cajal body proteins are still to be discovered.  we expected that the data-driven analyses would demonstrate that intra-nuclear compartments are functionally homogeneous and that their members would exhibit properties that enable their sorting. the collected large-scale protein data provides only partial evidence that the physical organization of the nucleus mirrors functional modularity. the current data set is far from completely annotated, suggesting that our methods for evaluating prediction accuracy are not robust. the error rates may be lower than what we have been able to conclude-ultimately we should instead rely on experimental validation. with the incomplete "trusted" data set, we fail to unambiguously identify proteins' intra-nuclear destination, suggesting that critical drivers behind of intra-nuclear translocation are yet to be identified. 
their gene ontology terms highlight their regulatory roles: splicing is confirmed to be a core activity of speckles and pml nuclear bodies house a range of proteins involved in dna repair. however, we are unable to unambiguously identify proteins' intra-nuclear destination, suggesting that critical drivers behind of intra-nuclear translocation are yet to be identified. prediction accuracy is highest for nucleoli and nuclear speckles. while the nucleolus and nuclear speckles have received more attention experimentally, the pml nuclear body and the cajal body are still incompletely characterized in terms of their roles and protein complement. we train support-vector machines to show that nuclear proteins contain discriminative sequence features that can be used to identify their intra-nuclear body associations. the trained models are also used to estimate the full protein complement of each nuclear body. cajal bodies, nucleoli, pml nuclear bodies, and nuclear speckles are morpohologically distinct intra-nuclear structures that dynamically respond to cellular cues. protein interactions are found primarily to link proteins in the nuclear speckles with proteins from other compartments. cell cycle expression data provide support for increased activity in nucleoli, nuclear speckles and pml nuclear bodies especially during s and g phases. the large-scale analysis of the mouse nuclear proteome sheds light on the functional organization of physically embodied intra-nuclear compartments. by collating recent experimentally verified data, we find that almost proteins in the mouse nuclear proteome are known to associate with one or more of the nuclear bodies. such nuclear bodies are hypothesized to play important regulatory roles, e.g. we observe partial support for the hypothesis that the physical organization of the nucleus mirrors functional modularity.
 cajal bodies, nucleoli, pml nuclear bodies, and nuclear speckles are morpohologically distinct intra-nuclear structures that dynamically respond to cellular cues. such nuclear bodies are hypothesized to play important regulatory roles, e.g. by sequestering and releasing transcription factors in a timely manner. while the nucleolus and nuclear speckles have received more attention experimentally, the pml nuclear body and the cajal body are still incompletely characterized in terms of their roles and protein complement.  by collating recent experimentally verified data, we find that almost proteins in the mouse nuclear proteome are known to associate with one or more of the nuclear bodies. their gene ontology terms highlight their regulatory roles: splicing is confirmed to be a core activity of speckles and pml nuclear bodies house a range of proteins involved in dna repair. we train support-vector machines to show that nuclear proteins contain discriminative sequence features that can be used to identify their intra-nuclear body associations. prediction accuracy is highest for nucleoli and nuclear speckles. the trained models are also used to estimate the full protein complement of each nuclear body. protein interactions are found primarily to link proteins in the nuclear speckles with proteins from other compartments. cell cycle expression data provide support for increased activity in nucleoli, nuclear speckles and pml nuclear bodies especially during s and g phases.  the large-scale analysis of the mouse nuclear proteome sheds light on the functional organization of physically embodied intra-nuclear compartments. we observe partial support for the hypothesis that the physical organization of the nucleus mirrors functional modularity. however, we are unable to unambiguously identify proteins' intra-nuclear destination, suggesting that critical drivers behind of intra-nuclear translocation are yet to be identified. 