BACKGROUND
alternative splicing is regulated in part by a network of signalling pathways which respond to extracellular stimuli  <cit> .
one molecule with a key role linking signalling and splicing is sam <dig> .
sam <dig> is the prototypic member of the star  family of rna-binding proteins  <cit> .
sam68-dependent splicing events impact upon important cellular decisions such as choices between cell survival and cell death  <cit> .
sam <dig> has also been recently shown to play an important role in neurogenesis through splicing regulation of specific pre-mrnas important for neural development  <cit> .
homozygous null sam <dig> mice have pleiotropic defects in bone morphogenesis, spermatogenesis and motor coordination suggesting widespread anatomical functions for the encoded protein  <cit> .
sam <dig> has been reported to be associated with a number of different proteins involved in rna processing, transcription, and cell signalling.
sam <dig> induces erk -mediated inclusion of cd <dig> variable exon v <dig> in response to ras activation  <cit> .
activation of cd <dig> exon v <dig> splicing by sam <dig> involves interactions with u2af <dig> to facilitate v <dig> exon definition  <cit> .
sam <dig> also interacts with the splicing repressor hnrnp a <dig>  <cit>  and nuclear transcriptional regulators  <cit> .
the amino acid sequence of sam <dig> protein contains several consensus motifs that mediate protein-protein and protein-rna interactions in response to different stimuli  <cit> .
in most somatic cells, sam <dig> protein is exclusively nuclear, but sam <dig> can also interact with cytoplasmic signalling molecules  <cit> .
sam <dig> protein has also been observed in the cytoplasm of secondary spermatocytes, where it is associated with polysomes and is involved in translational regulation  <cit> .
in cancer cells, sam <dig> protein exhibits a general nucleoplasmic distribution but is also concentrated within subnuclear structures called slm/sam <dig> nuclear bodies   <cit> .
although the exact function of snbs is unknown, they have been shown to contain some other splicing regulators, signalling components and nucleic acids.
although sam <dig> has a number of reported interacting protein partners, its major associated proteins are not yet known.
in this study, we searched by proteomics for the major interacting protein partners of nuclear sam <dig>  our data reveal hnrnp  l as a novel sam68-associated nuclear protein and a novel component of snbs.
RESULTS
hnrnp l is a novel sam68-interacting protein
sam <dig> protein is expressed at high levels in the nuclei of prostate cancer cells  <cit> .
we immunoprecipitated endogenous sam <dig> protein in nuclear extracts prepared from lncap cells.
western analysis confirmed that sam <dig> protein was efficiently immunoprecipitated by rabbit antisera specific for sam <dig>  but not by normal rabbit igg .
further analysis of the immunoprecipitates using sds-page followed by coomassie staining revealed a number of immunoprecipitated proteins, including a protein of ~ <dig> kda and a protein co-migrating with igg heavy chain , as well as a number of other distinct proteins.
peptide mass fingerprinting by maldi-tof  mass spectrometry was carried out on each of the immunoprecipitated proteins.
the mascot search engine was used to match peptides with statistically significant mowse  scores mapping across full-length protein sequences obtained from the swiss-prot database .
we identified the ~ <dig> kda band as sam <dig> , and other co-immunoprecipitated proteins as the known sam68-interacting proteins hnrnp a <dig>  <cit>  , hnrnp g  <cit>  , and hnrnp a2/b <dig>  <cit>  .
swiss-prot names and primary accession numbers for proteins identified by mascot searches.
p-values were calculated using the mowse algorithm.
nominal mass  and isoelectric point  were calculated using the integer mass of the most abundant isotope of each constituent element and the individual ionizing groups in each peptide, respectively.
in addition to the three previously known sam68-associated proteins, we also identified hnrnp l as a novel potential sam68-interacting protein partner .
by coomassie staining, the levels of co-precipitated hnrnp a <dig> and hnrnp g were similar to the level of immunoprecipitated sam <dig>  but there was considerably less co-immunoprecipitated hnrnp l .
hence, to independently confirm the novel sam68-hnrnp l interaction, both sam <dig> and hnrnp l proteins were separately immunoprecipitated in lncap cell nuclear extracts using their cognate antibodies prior to western analysis .
hnrnp l protein was efficiently immunoprecipitated by a monoclonal antibody specific for hnrnp l, and also co-immunoprecipitated by the antisera to sam <dig> .
likewise, sam <dig> protein was efficiently immunoprecipitated by its cognate antisera, and also co-immunoreprecipitated by the anti-hnrnp l antibody .
similar to previous reports  <cit> , we observed hnrnp l to migrate as a doublet, and both of these isoforms interacted with sam <dig> .
no or very weak immunoprecipitation was observed in the absence of either antibodies .
to further verify the novel sam68-hnrnp l detected interaction, hek <dig> cells were transiently transfected with expression constructs encoding flag-tagged sam <dig> and gst-tagged hnrnp l proteins.
anti-flag m <dig> agarose was used to immunoprecipitate flag-tagged proteins from hek <dig> cells, and co-immunoprecipitated gst fusion proteins were detected using antisera specific to gst.
consistent with the observed interactions of endogenous proteins, hnrnp l-gst was efficiently co-immunoprecipitated with sam68-flag , but could not be co-immunoprecipitated in cells transfected with an empty flag vector .
note that all immunoprecipitations of both endogenous  and flag-tagged proteins  were carried out in the presence of benzonase nuclease to ensure that the detected associations were mediated by protein-protein interactions and not bridged by nucleic acids.
sam <dig> and hnrnp l are both present in small protein complexes which are quantitatively associated with nucleic acids
sam <dig> is quantitatively associated with small nuclear protein complexes of a size compatible with either monomers or dimers  <cit> .
in order to investigate and compare the size ranges of any endogenous nuclear complexes containing hnrnp l protein, we analysed velocity gradient fractions of hek <dig> cell nuclear extracts prepared either without pretreatment or after prior micrococcal nuclease  treatment to eliminate any nucleic acid dependent complexes .
similarly to previously published data for sam <dig>  following mnase treatment, hnrnp l  protein was mainly present in small stable protein complexes  corresponding to the expected size of monomeric or dimeric molecular complexes .
also like sam <dig>  hnrnp l protein was quantitatively associated with nucleic acids since it migrated with much larger complexes without prior mnase digestion .
hnrnp l uniquely co-localises with sam <dig> as a novel component of snbs
within the nuclei of cancer cells, sam <dig> protein exhibits a general nucleoplasmic distribution but is also concentrated within distinct peri-nucleolar structures called snbs  <cit> .
we compared the localization of sam <dig> and hnrnp l proteins in cancer cell lines using indirect immunofluorescence .
confocal microscopy demonstrated that the endogenous sam <dig> and hnrnp l proteins directly overlapped within snbs in 100% of all cells examined.
in addition to snbs, further large pools of hnrnp l and sam <dig> proteins were seen in the general nucleoplasm, and in the case of hnrnp l, towards the nuclear periphery in saos- <dig> cells .
in parallel experiments, using the same combination of secondary antisera, no co-localisation was observed between sam <dig> protein  and sc35-containing splicing speckles  .
the distribution of sam <dig> and hnrnp l proteins was examined in three different cell lines  and found to be very similar suggesting that the hnrnp l association with snbs may be ubiquitous .
furthermore, the other identified sam68-associated proteins  identified in our proteomic screen exhibited diffuse subnuclear distributions and did not localise within snbs .
only a small number of molecules have been identified as snb components, and each of these share a role in cell signalling and/or rna processing.
hnrnp l has a closely-related homolog within the cell called hnrnp ll , which also regulates signal-dependent splicing.
we carried out further experiments to test if hnrnp ll protein would be also found within snbs.
surprisingly, these experiments indicated that hnrnp ll protein exhibits a nucleoplasmic distribution outside snbs .
the observed hnrnp ll distribution was punctate and somewhat reminiscent to that of splicing speckles.
we compared the localisation of hnrnp ll protein with sc <dig>  but found that the speckled distribution of hnrnp ll is in fact distinct from the sc35-containing splicing speckles .
to verify these localization findings, hela cells were transiently transfected with expression vectors encoding hnrnp l-gst, hnrnp ll-gst, or gst alone.
we then detected the localisation of the ectopically expressed proteins using antisera to detect the gst tag, and compared this distribution with that of endogenous sam <dig> protein .
consistent with the data described above, hnrnp l-gst but not hnrnp ll-gst, was found within snbs .
the gst moiety had no effect on protein localization, since gst alone was detected both within the nuclei and cytoplasm of transfected cells, and not within snbs .
sam <dig> and hnrnp l proteins associate in a cell line which exhibits a low frequency of snbs
the frequency of cells in a population containing snbs is cell line-dependent.
to test whether the association of sam <dig> and hnrnp l was dependent on the presence of snbs, we performed side-by-side immunoprecipitations in whole cell extracts obtained from lncap and nih3t <dig> cells   <cit> .
sam <dig> protein was efficiently immunoprecipitated by its cognate antisera from both these cell lines .
consistent with the interaction of sam <dig> and hnrnp l also occurring outside of snbs, virtually equal levels of hnrnp l protein was co-immunoprecipitated by the antisera to sam <dig> from nih3t <dig> cells as lncap cells .
notice that neither sam <dig> nor hnrnp l proteins were immunoprecipitated by normal rabbit igg or beads alone.
all immunoprecipitations were carried out in the presence of benzonase nuclease to confirm that the detected associations were mediated by protein-protein interactions and not bridged by nucleic acids.
the sam68-hnrnp l protein interaction does not significantly impact on splicing regulation of two known target exons
the above experiments indicate that sam <dig> and hnrnp l proteins interact and are both present within snbs.
although snbs contain some splicing regulators and rna, they do not detectably contain key spliceosome components  <cit> .
hence snbs are not thought to be sites for active pre-mrna splicing, which takes place in the general nucleoplasm  <cit> .
both sam <dig> and hnrnp l proteins are independently implicated in the regulation of distinct alternative splicing events during which exon inclusion can be either activated or repressed depending on the target transcript  <cit> .
although active splicing of sam <dig> and hnrnp l target pre-mrnas is unlikely to take place within snbs, we also detected significant nucleoplasmic populations of both sam <dig> and hnrnp l proteins outside snbs, and protein interactions between sam <dig> and hnrnp l occurred equally well in a cell line which exhibits a low frequency of snbs.
these observations raise the question as to whether the sam68-hnrnp l interaction might have a co-operative effect on pre-mrna splicing in the general nucleoplasm.
since ectopically expressed sam <dig> protein stimulates splicing inclusion of cd <dig> variable exon v <dig>  <cit> , and tjp <dig> exon  <dig> inclusion is repressed by hnrnp l  <cit> , we examined the effect of both sam <dig> and hnrnp l protein expression on these known target exons .
we firstly monitored splicing in hek <dig> cells transfected with a minigene containing cd <dig> variable exon v <dig> together with expression constructs encoding sam <dig>  hnrnp l or both .
ectopic expression of hnrnp l protein stimulated exon v <dig> inclusion albeit with a lower potency than sam <dig> .
we carried out similar experiments with a minigene containing tjp <dig> exon  <dig> .
due to the low levels of expression of tjp <dig> exon  <dig>  it was not easy to draw clear conclusions from this experiment, however ectopic expression of either hnrnp l or sam <dig> proteins alone appeared to repress exon  <dig> inclusion .
together, these experiments indicate that both sam <dig> and hnrnp l proteins can regulate the same pre-mrna splicing events in the same direction, with hnrnp l activating cd <dig> exon v <dig> inclusion like sam <dig>  and sam <dig> repressing tjp <dig> exon  <dig> inclusion like hnrnp l. importantly in either case, however, co-expression of hnrnp l and sam <dig> proteins did not have a significant additive effect on either tjp <dig> exon  <dig> skipping or cd <dig> exon v <dig> inclusion .
hence it seems unlikely that the general nucleoplasmic populations of hnrnp l and sam <dig> proteins co-operate in splicing control of these shared target exons, despite their observed interaction, localisations, and ability to regulate these target exons by themselves.
discussion
although sam <dig> has been associated with a number of proteins, our proteomics screen identified hnrnp a <dig>  hnrnp a2/b <dig>  hnrnp g and hnrnp l as its four major nuclear interacting protein partners in lncap cells.
the stoichiometry of the sam68-associated proteins recovered by immunoprecipitation, their spatial localisations, and analysis of endogenous complexes using velocity gradient centrifugation analysis  <cit>  are most consistent with a number of distinct small sam68-containing protein complexes , rather than a single large complex containing each of the identified sam68-interacting proteins.
like sam <dig> and the sam68-interactor hnrnp g, but not hnrnp a <dig>  hnrnp l protein was quantitatively associated with nucleic acids since it migrated with much larger complexes without prior mnase digestion before velocity gradient centrifugation.
hnrnp l protein has previously been described in discrete perinucleolar structures  <cit> , which we here now identify as snbs.
although the exact functions of snbs are not fully understood, they are known to contain other star proteins   <cit> , the splicing factor yt521b  <cit> , scaffold attachment factors safb <dig> and safb <dig>  <cit>  and the protein kinase brk/sik   <cit> .
snbs also contain nucleic acids including rna, and on heat-shock additionally recruit hnrnp a <dig> and other splicing factors including arginine-serine-rich  proteins  <cit> .
like sam <dig>  hnrnp l is implicated in coupling signaling and splicing: hnrnp l regulates signal-dependent splicing of cd <dig> exon v <dig>  <cit>  and strex   <cit> .
despite the spatial and physical associations of sam <dig> and hnrnp l detected in our study, sam <dig> and hnrnp l proteins did not either co-operate or antagonize each others' splicing activity on target exons.
in contrast, the generally nucleoplasmic sam68-associated protein hnrnp g potently inhibited sam68-mediated splicing of cd <dig> exon v <dig> .
our protein interaction analyses indicated that the interaction between sam <dig> and hnrnp l is independent of nucleic acids.
using a directed yeast two-hybrid system, a direct protein-protein interaction has been demonstrated between the star-family protein rslm- <dig> and hnrnp l  <cit> , suggesting, by analogy, that the sam68-hnrnp l interaction may also be direct.
however, we were unable to detect such an interaction by directed yeast two hybrid analysis .
amongst all the sam68-associated proteins we investigated in this study, the subnuclear distribution of hnrnp l protein is specifically within snbs.
consistent with the known nuclear functions of hnrnp l, snbs have been suggested as potential nuclear sites for the regulation of rna processing by signalling pathways.
however, we found that the homologous hnrnp ll protein which is also implicated in signal-dependent cd <dig> splicing  <cit> , did not localize to snbs.
hence snbs are not a default location for all nuclear rna-binding proteins involved in signaling and rna processing.
hnrnp ll protein lacks some proline- and glycine-rich regions present within the hnrnp l amino acid sequence, and by analogy with sam <dig>  it is possible that these motifs are required for interactions with signaling molecules and snb localization.
the distinct nuclear localization we observe for hnrnp ll may suggest somewhat diverged functions and so provide some explanation for the evolutionary maintenance of both hnrnp l and ll proteins within cells.
CONCLUSIONS
we have identified hnrnp l as a novel sam68-interacting protein partner and component of snbs.
despite hnrnp l and sam <dig> proteins also localizing to the general nucleoplasm, where pre-mrna splicing occurs, we did not observe a role for the novel sam68-hnrnp l protein interaction on splicing of known target exons for these rna-binding proteins.
the spatial localization of hnrnp l protein within snbs further implicates a role for these organelles in coupling signaling to rna processing.

 alternative splicing is regulated in part by a network of signalling pathways which respond to extracellular stimuli. one molecule with a key role linking signalling and splicing is sam. sam is the prototypic member of the star family of rna-binding proteins. sam68-dependent splicing events impact upon important cellular decisions such as choices between cell survival and cell death. sam has also been recently shown to play an important role in neurogenesis through splicing regulation of specific pre-mrnas important for neural development. homozygous null sam mice have pleiotropic defects in bone morphogenesis, spermatogenesis and motor coordination suggesting widespread anatomical functions for the encoded protein. sam has been reported to be associated with a number of different proteins involved in rna processing, transcription, and cell signalling. sam induces erk -mediated inclusion of cd variable exon v in response to ras activation. activation of cd exon v splicing by sam involves interactions with u2af to facilitate v exon definition. sam also interacts with the splicing repressor hnrnp a and nuclear transcriptional regulators. the amino acid sequence of sam protein contains several consensus motifs that mediate protein-protein and protein-rna interactions in response to different stimuli. in most somatic cells, sam protein is exclusively nuclear, but sam can also interact with cytoplasmic signalling molecules. sam protein has also been observed in the cytoplasm of secondary spermatocytes, where it is associated with polysomes and is involved in translational regulation. in cancer cells, sam protein exhibits a general nucleoplasmic distribution but is also concentrated within subnuclear structures called slm/sam nuclear bodies. although the exact function of snbs is unknown, they have been shown to contain some other splicing regulators, signalling components and nucleic acids. although sam has a number of reported interacting protein partners, its major associated proteins are not yet known. in this study, we searched by proteomics for the major interacting protein partners of nuclear sam our data reveal hnrnp l as a novel sam68-associated nuclear protein and a novel component of snbs.  hnrnp l is a novel sam68-interacting protein sam protein is expressed at high levels in the nuclei of prostate cancer cells. we immunoprecipitated endogenous sam protein in nuclear extracts prepared from lncap cells. western analysis confirmed that sam protein was efficiently immunoprecipitated by rabbit antisera specific for sam but not by normal rabbit igg. further analysis of the immunoprecipitates using sds-page followed by coomassie staining revealed a number of immunoprecipitated proteins, including a protein of ~ kda and a protein co-migrating with igg heavy chain, as well as a number of other distinct proteins. peptide mass fingerprinting by maldi-tof mass spectrometry was carried out on each of the immunoprecipitated proteins. the mascot search engine was used to match peptides with statistically significant mowse scores mapping across full-length protein sequences obtained from the swiss-prot database. we identified the ~ kda band as sam, and other co-immunoprecipitated proteins as the known sam68-interacting proteins hnrnp a, hnrnp g, and hnrnp a2/b. swiss-prot names and primary accession numbers for proteins identified by mascot searches. p-values were calculated using the mowse algorithm. nominal mass and isoelectric point were calculated using the integer mass of the most abundant isotope of each constituent element and the individual ionizing groups in each peptide, respectively. in addition to the three previously known sam68-associated proteins, we also identified hnrnp l as a novel potential sam68-interacting protein partner. by coomassie staining, the levels of co-precipitated hnrnp a and hnrnp g were similar to the level of immunoprecipitated sam but there was considerably less co-immunoprecipitated hnrnp l. hence, to independently confirm the novel sam68-hnrnp l interaction, both sam and hnrnp l proteins were separately immunoprecipitated in lncap cell nuclear extracts using their cognate antibodies prior to western analysis. hnrnp l protein was efficiently immunoprecipitated by a monoclonal antibody specific for hnrnp l, and also co-immunoprecipitated by the antisera to sam. likewise, sam protein was efficiently immunoprecipitated by its cognate antisera, and also co-immunoreprecipitated by the anti-hnrnp l antibody. similar to previous reports, we observed hnrnp l to migrate as a doublet, and both of these isoforms interacted with sam. no or very weak immunoprecipitation was observed in the absence of either antibodies. to further verify the novel sam68-hnrnp l detected interaction, hek cells were transiently transfected with expression constructs encoding flag-tagged sam and gst-tagged hnrnp l proteins. anti-flag m agarose was used to immunoprecipitate flag-tagged proteins from hek cells, and co-immunoprecipitated gst fusion proteins were detected using antisera specific to gst. consistent with the observed interactions of endogenous proteins, hnrnp l-gst was efficiently co-immunoprecipitated with sam68-flag, but could not be co-immunoprecipitated in cells transfected with an empty flag vector. note that all immunoprecipitations of both endogenous and flag-tagged proteins were carried out in the presence of benzonase nuclease to ensure that the detected associations were mediated by protein-protein interactions and not bridged by nucleic acids. sam and hnrnp l are both present in small protein complexes which are quantitatively associated with nucleic acids sam is quantitatively associated with small nuclear protein complexes of a size compatible with either monomers or dimers. in order to investigate and compare the size ranges of any endogenous nuclear complexes containing hnrnp l protein, we analysed velocity gradient fractions of hek cell nuclear extracts prepared either without pretreatment or after prior micrococcal nuclease treatment to eliminate any nucleic acid dependent complexes. similarly to previously published data for sam following mnase treatment, hnrnp l protein was mainly present in small stable protein complexes corresponding to the expected size of monomeric or dimeric molecular complexes. also like sam hnrnp l protein was quantitatively associated with nucleic acids since it migrated with much larger complexes without prior mnase digestion. hnrnp l uniquely co-localises with sam as a novel component of snbs within the nuclei of cancer cells, sam protein exhibits a general nucleoplasmic distribution but is also concentrated within distinct peri-nucleolar structures called snbs. we compared the localization of sam and hnrnp l proteins in cancer cell lines using indirect immunofluorescence. confocal microscopy demonstrated that the endogenous sam and hnrnp l proteins directly overlapped within snbs in 100% of all cells examined. in addition to snbs, further large pools of hnrnp l and sam proteins were seen in the general nucleoplasm, and in the case of hnrnp l, towards the nuclear periphery in saos- cells. in parallel experiments, using the same combination of secondary antisera, no co-localisation was observed between sam protein and sc35-containing splicing speckles. the distribution of sam and hnrnp l proteins was examined in three different cell lines and found to be very similar suggesting that the hnrnp l association with snbs may be ubiquitous. furthermore, the other identified sam68-associated proteins identified in our proteomic screen exhibited diffuse subnuclear distributions and did not localise within snbs. only a small number of molecules have been identified as snb components, and each of these share a role in cell signalling and/or rna processing. hnrnp l has a closely-related homolog within the cell called hnrnp ll, which also regulates signal-dependent splicing. we carried out further experiments to test if hnrnp ll protein would be also found within snbs. surprisingly, these experiments indicated that hnrnp ll protein exhibits a nucleoplasmic distribution outside snbs. the observed hnrnp ll distribution was punctate and somewhat reminiscent to that of splicing speckles. we compared the localisation of hnrnp ll protein with sc but found that the speckled distribution of hnrnp ll is in fact distinct from the sc35-containing splicing speckles. to verify these localization findings, hela cells were transiently transfected with expression vectors encoding hnrnp l-gst, hnrnp ll-gst, or gst alone. we then detected the localisation of the ectopically expressed proteins using antisera to detect the gst tag, and compared this distribution with that of endogenous sam protein. consistent with the data described above, hnrnp l-gst but not hnrnp ll-gst, was found within snbs. the gst moiety had no effect on protein localization, since gst alone was detected both within the nuclei and cytoplasm of transfected cells, and not within snbs. sam and hnrnp l proteins associate in a cell line which exhibits a low frequency of snbs the frequency of cells in a population containing snbs is cell line-dependent. to test whether the association of sam and hnrnp l was dependent on the presence of snbs, we performed side-by-side immunoprecipitations in whole cell extracts obtained from lncap and nih3t cells. sam protein was efficiently immunoprecipitated by its cognate antisera from both these cell lines. consistent with the interaction of sam and hnrnp l also occurring outside of snbs, virtually equal levels of hnrnp l protein was co-immunoprecipitated by the antisera to sam from nih3t cells as lncap cells. notice that neither sam nor hnrnp l proteins were immunoprecipitated by normal rabbit igg or beads alone. all immunoprecipitations were carried out in the presence of benzonase nuclease to confirm that the detected associations were mediated by protein-protein interactions and not bridged by nucleic acids. the sam68-hnrnp l protein interaction does not significantly impact on splicing regulation of two known target exons the above experiments indicate that sam and hnrnp l proteins interact and are both present within snbs. although snbs contain some splicing regulators and rna, they do not detectably contain key spliceosome components. hence snbs are not thought to be sites for active pre-mrna splicing, which takes place in the general nucleoplasm. both sam and hnrnp l proteins are independently implicated in the regulation of distinct alternative splicing events during which exon inclusion can be either activated or repressed depending on the target transcript. although active splicing of sam and hnrnp l target pre-mrnas is unlikely to take place within snbs, we also detected significant nucleoplasmic populations of both sam and hnrnp l proteins outside snbs, and protein interactions between sam and hnrnp l occurred equally well in a cell line which exhibits a low frequency of snbs. these observations raise the question as to whether the sam68-hnrnp l interaction might have a co-operative effect on pre-mrna splicing in the general nucleoplasm. since ectopically expressed sam protein stimulates splicing inclusion of cd variable exon v, and tjp exon inclusion is repressed by hnrnp l, we examined the effect of both sam and hnrnp l protein expression on these known target exons. we firstly monitored splicing in hek cells transfected with a minigene containing cd variable exon v together with expression constructs encoding sam hnrnp l or both. ectopic expression of hnrnp l protein stimulated exon v inclusion albeit with a lower potency than sam. we carried out similar experiments with a minigene containing tjp exon. due to the low levels of expression of tjp exon it was not easy to draw clear conclusions from this experiment, however ectopic expression of either hnrnp l or sam proteins alone appeared to repress exon inclusion. together, these experiments indicate that both sam and hnrnp l proteins can regulate the same pre-mrna splicing events in the same direction, with hnrnp l activating cd exon v inclusion like sam and sam repressing tjp exon inclusion like hnrnp l. importantly in either case, however, co-expression of hnrnp l and sam proteins did not have a significant additive effect on either tjp exon skipping or cd exon v inclusion. hence it seems unlikely that the general nucleoplasmic populations of hnrnp l and sam proteins co-operate in splicing control of these shared target exons, despite their observed interaction, localisations, and ability to regulate these target exons by themselves. discussion although sam has been associated with a number of proteins, our proteomics screen identified hnrnp a hnrnp a2/b hnrnp g and hnrnp l as its four major nuclear interacting protein partners in lncap cells. the stoichiometry of the sam68-associated proteins recovered by immunoprecipitation, their spatial localisations, and analysis of endogenous complexes using velocity gradient centrifugation analysis are most consistent with a number of distinct small sam68-containing protein complexes, rather than a single large complex containing each of the identified sam68-interacting proteins. like sam and the sam68-interactor hnrnp g, but not hnrnp a hnrnp l protein was quantitatively associated with nucleic acids since it migrated with much larger complexes without prior mnase digestion before velocity gradient centrifugation. hnrnp l protein has previously been described in discrete perinucleolar structures, which we here now identify as snbs. although the exact functions of snbs are not fully understood, they are known to contain other star proteins, the splicing factor yt521b, scaffold attachment factors safb and safb and the protein kinase brk/sik. snbs also contain nucleic acids including rna, and on heat-shock additionally recruit hnrnp a and other splicing factors including arginine-serine-rich proteins. like sam hnrnp l is implicated in coupling signaling and splicing: hnrnp l regulates signal-dependent splicing of cd exon v and strex. despite the spatial and physical associations of sam and hnrnp l detected in our study, sam and hnrnp l proteins did not either co-operate or antagonize each others' splicing activity on target exons. in contrast, the generally nucleoplasmic sam68-associated protein hnrnp g potently inhibited sam68-mediated splicing of cd exon v. our protein interaction analyses indicated that the interaction between sam and hnrnp l is independent of nucleic acids. using a directed yeast two-hybrid system, a direct protein-protein interaction has been demonstrated between the star-family protein rslm- and hnrnp l, suggesting, by analogy, that the sam68-hnrnp l interaction may also be direct. however, we were unable to detect such an interaction by directed yeast two hybrid analysis. amongst all the sam68-associated proteins we investigated in this study, the subnuclear distribution of hnrnp l protein is specifically within snbs. consistent with the known nuclear functions of hnrnp l, snbs have been suggested as potential nuclear sites for the regulation of rna processing by signalling pathways. however, we found that the homologous hnrnp ll protein which is also implicated in signal-dependent cd splicing, did not localize to snbs. hence snbs are not a default location for all nuclear rna-binding proteins involved in signaling and rna processing. hnrnp ll protein lacks some proline- and glycine-rich regions present within the hnrnp l amino acid sequence, and by analogy with sam it is possible that these motifs are required for interactions with signaling molecules and snb localization. the distinct nuclear localization we observe for hnrnp ll may suggest somewhat diverged functions and so provide some explanation for the evolutionary maintenance of both hnrnp l and ll proteins within cells.  we have identified hnrnp l as a novel sam68-interacting protein partner and component of snbs. despite hnrnp l and sam proteins also localizing to the general nucleoplasm, where pre-mrna splicing occurs, we did not observe a role for the novel sam68-hnrnp l protein interaction on splicing of known target exons for these rna-binding proteins. the spatial localization of hnrnp l protein within snbs further implicates a role for these organelles in coupling signaling to rna processing. 
although ectopic expression of hnrnp l and sam proteins independently affected splicing of cd variable exon v and tjp exon minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons. the interaction between sam and hnrnp l proteins was observed in a cell line which exhibits low frequency of snbs suggesting that this association also takes place outside snbs. we used proteomics to search for the major interacting protein partners of nuclear sam in addition to sam itself and known sam68-associated proteins, we identified hnrnp l as a novel sam68-interacting protein partner. hnrnp l protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnrnp l spatially co-localised with sam as a novel component of snbs and was also observed within the general nucleoplasm. localisation within snbs was highly specific to hnrnp l and was not shared by the closely-related hnrnp ll protein, nor any of the other sam68-interacting proteins we identified by proteomics. here we identify hnrnp l as a novel snb component. we show that, compared with other identified sam68-associated hnrnp proteins and hnrnp ll, this co-localisation within snbs is specific to hnrnp l. our data suggest that the novel sam68-hnrnp l protein interaction may have a distinct role within snbs. nuclear sam protein is concentrated within subnuclear organelles called slm/sam nuclear bodies, which also contain some other splicing regulators, signalling components and nucleic acids. sam is the prototypic member of the star family of rna-binding proteins, which regulate splicing in response to signalling cascades. active pre-mrna splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells.
 active pre-mrna splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. splicing decisions are controlled by networks of nuclear rna-binding proteins and their target sequences, sometimes in response to signalling pathways. sam is the prototypic member of the star family of rna-binding proteins, which regulate splicing in response to signalling cascades. nuclear sam protein is concentrated within subnuclear organelles called slm/sam nuclear bodies, which also contain some other splicing regulators, signalling components and nucleic acids.  we used proteomics to search for the major interacting protein partners of nuclear sam in addition to sam itself and known sam68-associated proteins, we identified hnrnp l as a novel sam68-interacting protein partner. hnrnp l protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnrnp l spatially co-localised with sam as a novel component of snbs and was also observed within the general nucleoplasm. localisation within snbs was highly specific to hnrnp l and was not shared by the closely-related hnrnp ll protein, nor any of the other sam68-interacting proteins we identified by proteomics. the interaction between sam and hnrnp l proteins was observed in a cell line which exhibits low frequency of snbs suggesting that this association also takes place outside snbs. although ectopic expression of hnrnp l and sam proteins independently affected splicing of cd variable exon v and tjp exon minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons.  here we identify hnrnp l as a novel snb component. we show that, compared with other identified sam68-associated hnrnp proteins and hnrnp ll, this co-localisation within snbs is specific to hnrnp l. our data suggest that the novel sam68-hnrnp l protein interaction may have a distinct role within snbs. 