BACKGROUND
the biogenesis of most spliceosomal small nuclear ribonucleoproteins  is complicated and requires both cytoplasmic and nuclear maturation steps  <cit> .
for example, the spliceosomal small nuclear rnas  of ul, u <dig>  u <dig> and u <dig> snrnps are synthesized by rna polymerase ii and may traffic through specific subnuclear domains before being exported to the cytoplasm  <cit> .
in the cytoplasm, a septet of sm proteins  binds the sm motif of the snrna under the control of the survival of motor neurons  protein complex.
mutations in the smn protein cause the neurodegenerative disorder spinal muscular atrophy  <cit> .
after the sm core has been assembled onto the snrna, the snrna is subject to further processing, followed by import back into the nucleus; again with the help of the smn complex  <cit> .
upon nuclear re-entry, newly assembled ul, u <dig>  u <dig> and u <dig> snrnps first localize to a subnuclear domain known as the cajal body  <cit> .
in the cajal body , the snrna component of the snrnp is subjected to pseudouridine-base and 2'-0-methyl sugar-modifications that are guided by small cb-specific rnas   <cit> .
these modifications are crucial for proper pre-mrna splicing in vivo  <cit> .
after their modification in the cb, snrnps localize to speckles, where they are stored, or perichromatin fibrils, where splicing occurs concurrently with transcription  <cit> .
unlike ul, u <dig>  u <dig> and u <dig> snrnas, maturation of the rna polymerase iii-transcribed u <dig> snrna does not include a cytoplasmic phase and may take place in the nucleolus and the cb  <cit> .
the u <dig> snrnp, which is required for histone pre-mrna 3'-end processing  <cit> , is assembled in manner similar to that observed for ul, u <dig>  u <dig> and u <dig> snrnps, including a cytoplasmic phase  <cit> .
however, u <dig> snrna has a noncanonical sm binding site and thus recruits a different type of sm core compared to that which binds ul, u <dig>  u <dig> and u <dig> snrna  <cit> .
like the spliceosomal snrnps, the u <dig> snrnp is enriched within cbs  <cit> .
cbs have been shown to move in an atp dependent manner  <cit>  as well as associate with various gene loci, including snrna and histone gene clusters  <cit> .
consequently, cbs may provide a platform upon which a feedback regulatory mechanism for snrnp and histone biogenesis takes place  <cit> .
the mechanisms by which snrnps are targeted to and released from the cb are unknown.
one possibility is that a factor within the cb interacts with nascent snrnps and facilitates their modification.
another factor may displace snrnps from the cb, allowing for their subsequent localization in speckles and perichromatin fibrils.
the cb marker protein coilin may play a role in the targeting of snrnps to cbs.
removal of coilin in xenopus by immunodepletion decreases snrnp levels in the amphibian equivalent of the cb  <cit> .
characterization of coilin knockout mice has revealed that they are viable on an outbred mouse strain, but have significant viability and fertility defects on inbred strains  <cit>  .
cell lines derived from coilin knockout mice lack canonical cbs in which snrnps are enriched  <cit> .
however, add-back experiments demonstrate that typical cbs, containing snrnps, can be reformed upon the addition of coilin  <cit> .
furthermore, coilin can interact directly with several sm proteins of snrnps  <cit>  .
taken together, these data indicate that, while not an essential protein, coilin is important for proper cb formation.
functional cbs may allow for the efficient coordination of the nuclear steps of snrnp biogenesis.
another protein that requires coilin for its localization to cbs is smn  <cit> .
coilin directly interacts with smn via several arginine/glycine  dipeptide repeats in coilin  <cit> .
the arginines within this rg box motif are symmetrically dimethylated  <cit> , resulting in an increased affinity for smn  <cit> .
in most cell lines and tissues, smn localizes to the cytoplasm and cbs  <cit> .
however, smn in some cell lines and fetal tissue localizes to discrete nuclear structures termed "gems"   <cit> .
the presence of gems correlates with a decrease in coilin methylation  <cit> .
although the nuclear role of smn is not well understood, it is possible that smn escorts nascent snrnps to the cb  <cit> .
interestingly, coilin can compete with sm proteins for binding sites on smn  <cit> , indicating that locally high concentrations of coilin within the cb may serve to displace snrnps from the smn complex.
the interplay between smn, snrnps and coilin may therefore regulate snrnp accumulation within the cb.
we set out to determine if we could identify factors that control the departure of nascent snrnps from the cb.
in this report, we demonstrate that an isoform of the human mitochondrial tim <dig>  tim50a, localizes to speckles and interacts with snrnps, smn and coilin.
human tim <dig>  which has phosphatase activity, is a component of the mitochondrial translocator and regulates mitochondrial integrity and cell death  <cit> .
the protein sequence of tim <dig> and tim50a are identical, with the important exception that tim50a contains an alternative translational start sequence that adds  <dig> aa to its n-terminus relative to tim <dig>  competition binding experiments show that smb' does not compete with smn for tim50a binding sites, but smb' does reduce coilin interaction with tim50a.
furthermore, smn and coilin compete for tim50a binding sites and tim50a forms a more efficient complex with snrnps in vivo compared to tim <dig>  based on these results, we propose that tim50a is involved in the regulation of snrnp biogenesis and possibly the activity of nuclear smn.
RESULTS
isolation of tim50a
given the central role of coilin in the formation and composition of cbs, especially in its ability to recruit the smn complex and snrnps, we wanted to identify proteins that interact with coilin and possibly regulate smn and snrnp localization in the cb.
towards this end, we conducted a yeast two-hybrid screen with coilin as bait.
a human brain cdna library was chosen for this screen because we are interested in assessing cb protein dynamics in neuronal tissue, which is the cell type primarily affected in spinal muscular atrophy.
the c-terminal  <dig> aa of coilin, which interacts with smn and sm proteins, was the bait for the screen  <cit>  .
consequently, other proteins that bind c <dig> may regulate the interplay between coilin/smn and coilin/sm and thus play a role in cb dynamics.
several preys were recovered from the screen, one of which was termed cap <dig>  sequencing of this prey revealed that it contained the partial coding sequence for a protein known as tim <dig>  since tim <dig> localizes to the mitochondria and cytoplasm  <cit> , the relevance of cap <dig> interaction with coilin, a nuclear protein, was not initially clear.
however, upon conducting est  database searches, we found that there are two possible translational starts for the timm <dig> gene product.
the majority of ests terminate slightly upstream of the published translational start sequence of tim <dig> , suggesting that tim <dig> is the major product of the timm <dig> gene.
however, three ests, all from cancer cells lines, demonstrate that an alternative translational start sequence can be utilized which would add  <dig> aa to the n-terminus of the tim <dig> protein.
we have named this protein tim50a .
importantly, a putative nuclear localization signal   <cit>  is found within this region, indicating that tim50a, unlike tim <dig>  may reside in the nucleus.
to verify the existence of tim50a in other cell lines and to obtain a general idea as to the abundance of this transcript relative to the tim <dig> isoform, we performed nested pcr with primers specific to tim50a on cdnas from four additional cancer cell lines: a- <dig>  nih: ovcar- <dig>  mda-mb- <dig> and hct- <dig>  another primer set was used in a standard  pcr reaction to amplify a region common to both tim50a and tim <dig> .
as shown in figure 1c , a standard pcr reaction  using primers that can bind both tim50a and tim <dig>  generate a product from all of the cell lines tested.
in contrast, standard pcr reactions using tim50a-specific primers  fail to yield a product from cell line cdnas, but successfully generate a product using a positive control plasmid template .
however, nested pcr  reveals that two of the four cell lines, a- <dig> and nih: ovcar- <dig>  contain the tim50a transcript , as monitored by sequencing of the product.
the lower band in the nih: ovcar- <dig> reaction is from an unrelated transcript.
therefore, while tim50a mrna is not nearly as abundant as the tim <dig> message, this species is present in at least four different cell lines .
cellular localization of tim50a
to test if tim50a is a nuclear protein, we obtained an est encoding the additional residues and cloned the entire tim50a coding sequence into a gfp expression vector.
we also made a gfp fusion to the truncated tim <dig> protein isolated from the two-hybrid screen  as well as gfp fusions to various mutants of tim50a.
the localization of these proteins, and gfp fused to the mitochondrial tim <dig> isoform, was monitored after transient transfection into hela cells .
in contrast to tim <dig>  gfp-tim50a is exclusively nuclear with speckle-like accumulations .
to confirm that the nuclear accumulations are indeed speckles, in which snrnps are stored, we stained the cells with antibodies against sm proteins .
there is overlap between tim50a and snrnps in speckles , as shown by the yellow-orange signal in the merged image .
we do not detect tim50a in cajal bodies , indicating that the association of tim50a with cbs or coilin is weak or transient.
alternatively, coilin may associate with tim50a in the nucleoplasm.
indeed, while coilin is highly enriched in the cb, the majority  of the protein is diffusely localized throughout the nucleoplasm  <cit> .
we observed identical localization patterns for tim50a upon fusion to a smaller myc-tag , suggesting that the gfp-tag does not affect localization.
the diffuse cytoplasmic and nuclear localization of gfp-cap <dig> and gfp-tim50a demonstrate that the region containing the putative nls is necessary for nuclear localization .
tim50a mutants that retain the putative nls display nuclear accumulation.
for example, an internal deletion of tim50a  is found in a speckled nuclear pattern while the first  <dig> aa  is nuclear and nucleolar.
tim50a localization to speckles is therefore contingent upon the nls and residues found between aa  <dig> and  <dig>
actinomycin d redistribution of tim50a
the transcription inhibitor actinomycin d can be used to partially characterize the interaction of proteins in the nucleus.
this is because actinomycin d inhibits transcription and causes the reorganization of splicing factors and cajal bodies  <cit> .
in particular, speckles enlarge and cbs are relocalized to the periphery of the nucleolus in the presence of actinomycin d  <cit> .
if tim50a were an integral part of speckles, then we would suspect that this protein would be found in enlarged speckles when cells are treated with actinomycin d. however, in the presence of actinomycin d, tim50a no longer localizes with snrnps in speckles but is instead found in discrete structures .
this is evident in the merged image, which shows separate green  and red  signals with little overlap.
in contrast, non-treated cells display overlap between tim50a and snrnps, resulting in a yellow-orange signal in the merged image .
we tested whether tim50a in actinomycin d treated cells localized to paraspeckles, another subnuclear domain of unknown function  <cit> .
no colocalization of tim50a with the paraspeckle marker protein, psp <dig>  was observed .
taken together, the localization data indicate that tim50a is likely to have a different function compared to tim <dig>  possibly in the regulation of cb activity or snrnp biogenesis.
however tim50a is not an integral component of speckles given the separate localization pattern of tim50a and snrnps in actinomycin d treated cells.
direct interaction of tim50a with coilin, smb' and smn
the two-hybrid screen suggests that tim <dig> isoforms may interact with coilin.
to prove this, we conducted gst-pulldown assays using bacterially purified t7-tagged coilin and tim50a fused to gst.
coilin is not recovered over beads fused to gst alone, but is recovered over gst-tim50a beads .
we next tested if tim50a would interact with the c-terminal  <dig> aa of coilin , which is this same fragment of coilin used to isolate cap <dig> in the two-hybrid screen.
in agreement with the two-hybrid results, soluble tim50a is recovered over gst-c <dig> beads, but not by gst alone .
since tim50a localizes to speckles, we were interested in assessing if tim50a interacts with sm proteins of snrnps.
using smb' as a representative of sm proteins, we conducted gst-pulldown experiments with a variety of gst-tim50a proteins.
consistent with the localization data, smb' directly interacts with gst-tim50a .
smb' also associates with two mutants of tim50a, cap <dig> and tim50sac.
based on the binding results, we suspect that the interaction domain for smb' on tim50a lies somewhere between residues  <dig> and  <dig> of tim50a, although we cannot exclude the role of the residues at the extreme c-terminus of tim50a .
given that tim50a localizes to speckles and directly interacts with coilin and smb', we next tested if tim50a could interact with another protein crucial for snrnp formation, smn.
as shown in figure 6d, smn binds to the gst-tim50a proteins but not to gst alone.
we also examined if the isoform known to localize in mitochondria, tim <dig>  also interacts with the coilin fragment c <dig> as found for tim50a.
purified full-length tim <dig> does indeed interact specifically with the coilin fragment as shown in figure 6e.
from these in vitro binding studies, we conclude that coilin, smb' and smn can interact with both tim50a and tim <dig> isoforms, but the interaction of these proteins in vivo is likely regulated by their subcellular localization.
smb' and smn compete with coilin for tim50a binding sites
the ability of tim50a to bind coilin, smb' and smn raises the interesting possibility that this protein may regulate the dynamics of cb proteins.
to test this hypothesis, we conducted competition gst-pulldown experiments using gst-tim50asac.
this protein was used instead of gst-tim50a  because it is more easily purified compared to gst-tim50a and has similar binding capabilities .
for the first experiment, we tested if smn and smb' vie for the same binding sites on tim50a.
this was accomplished by incubating a fixed amount of gst-tim50asac with a constant amount of smn and, in separate reactions, an increasing amount of smb'.
despite binding a large amount of smb' , the level of smn recovered by the gst-tim50asac beads does not change .
we conclude that tim50a has separate binding sites for smn and smb' and that one protein does not preclude the other from binding.
we then tested if coilin and smb' compete for binding sites on tim50a.
for this experiment, we incubated a fixed amount of gst-tim50asac with a constant amount of a coilin fragment  and, in separate reactions, an increasing amount of smb'.
as shown in figure 7b, increasing the level of smb' decreases the recovery of the coilin fragment  by gst-tim50asac .
thus coilin and smb' compete for binding sites on tim50a.
finally we monitored if coilin and smn compete for the same binding sites on tim50a.
this experiment was conducted with a fixed amount of gst-tim50asac and a constant amount of smn with increasing levels of the coilin fragment .
the binding of the respective proteins to the tim50a substrate in the absence of competitor  is shown in lane  <dig>  for the coilin fragment and lane  <dig>  for smn.
in reactions with both soluble proteins, as the level of coilin fragment  is increased, the amount of smn recovered by the tim50a beads is reduced .
these data are consistent with a competition for tim50a binding sites by smn and coilin.
given its protein interaction potential, we hypothesize that tim50a may affect cb function by interacting with proteins that localize to this domain.
one possible scenario is that tim50a displaces snrnps from the cb and facilitates their translocation to speckles.
tim50a may also disrupt the interaction between coilin and smn, allowing for smn to leave the cb.
tim50a interacts with smn and snrnps in vivo
to corroborate the direct interaction data, we tested if tim50a could associate with smn in cells.
for these experiments, we transfected hela cells with empty gfp vector, gfp-tagged full-length tim50a or gfp fused to the n-terminal  <dig> aa of tim50a.
after incubation, lysates were made and subjected to immunoprecipitation with anti-gfp antibodies, followed by western blotting with anti-smn antibodies.
as shown in figure 8a , no smn is co-immunoprecipitated with gfp alone but smn is recovered by gfp-tim50a.
only a faint smn signal is observed in the gfp-tim50a reaction , suggesting that this fragment does not contribute significantly to the ability of smn to bind tim50a.
reprobing of this same blot with anti-gfp  demonstrates that while a large amount of gfp and gfp-tim50a were immunoprecipitated, little smn was recovered.
in contrast, the amount of immunoprecipitated gfp-tim50a is less than that obtained for gfp and gfp-tim50a, yet more smn was recovered.
thus tim50a can form a complex with smn in cells.
given that tim50a localizes to speckles, in which snrnps are stored, and directly interacts with a subunit of the sm core, it is likely that tim50a will interact with intact snrnps.
however, the in vitro binding data indicate that the mitochondrial isoform, tim <dig>  also has the potential to interact with snrnps, which are known to have a cytoplasmic phase.
to test this, we conducted a co-immunoprecipitation assay on hela cells transfected with empty gfp vector or gfp fused to tim50a or tim <dig>  lysates were generated and subject to incubation with the anti-sm antibody y <dig>  after sds-page and western transfer, the blot was probed with anti-gfp antibodies to monitor the association of gfp-tim50a or gfp-tim <dig> with snrnps.
as shown in figure 8b, a signal corresponding to gfp-tim50a is present in the y <dig> immunoprecipitation reaction .
in contrast, less tim <dig> is co-immunoprecipitated by y <dig>  compared to tim50a.
a gfp-alone signal is not observed in the y <dig> reaction , demonstrating the specificity of the reaction.
therefore, in vivo, tim50a appears to be more efficient in snrnp interaction than tim <dig>  although we have not excluded the possibility that the gfp-tag may adversely affect tim <dig> interactions.
to address this point, tim50a and tim <dig> proteins were fused to smaller myc-tags, expressed in hela cells and tested for interaction with an immobilized sm protein, smb'.
both proteins bind gst-smb', indicating that the gfp-tag may indeed contribute to the reduced interaction of tim <dig> with snrnps .
alternatively, the faint tim <dig> signal observed in the y <dig> co-immunoprecipitation reaction  may be an artifact due to the overexpression of gfp-tim <dig> and subsequent nuclear localization, as shown in figure  <dig>  in support of this thought, immunofluorescence analysis of endogenous tim <dig> demonstrates that this protein is predominantly cytoplasmic with little nuclear accumulation  <cit> .
given that tim50a is a nuclear protein, we speculate that tim50a interactions may regulate the nuclear activity of smn and trafficking of snrnps, possibly by altering the dynamics of the association between smn, coilin and snrnps.
additionally, it is plausible that cytoplasmic smn can interact with tim <dig> before mitochondrial import.
CONCLUSIONS
the cellular localization and protein interaction characteristics of tim50a suggest that this isoform of tim <dig> may play a role in snrnp biogenesis.
based on the abundance of ests, it is likely that tim <dig>  not tim50a, is the major product of the timm <dig> gene.
however, the presence of tim50a ests from cancer cell lines suggests that this protein may be induced in cellular transformation.
it is known that cancer cells contain cbs, while some normal cells and tissues do not  <cit> .
for example, normal adult lung tissue does not contain cbs, but lung cancer cells do.
since cbs play a role in snrnp biogenesis, it is likely that the increase of cbs in transformed cells reflects on the heightened demand in splicing requirements for upregulated genes that participate in the establishment and/or maintenance of the altered cellular physiology  <cit> .
the induction of cbs may also facilitate the production of telomerase, the rna component of which localizes to cbs  <cit> .
in addition to cancer cells neuronal cells contain robust cbs  <cit> , possibly because high levels of snrnps are necessary to process the abundance of messages subject to alternative splicing in neuronal tissue  <cit> .
therefore, tim50a expression may be limited to tissue with high demands in snrnp metabolism.
we are currently developing an antibody to the unique n-terminus of tim50a in order to explore this possibility.
additionally, experiments are underway to verify the role of tim50a in snrnp biogenesis.
in particular, we are interesting in determining if tim50a is involved in the liberation of snrnps from the cb after they have been modified.
the functional consequence of the interaction between tim50a and smn is also being investigated.
finally we note that tim50a contains the phosphatase domain shown to be functional in the tim <dig> isoform  <cit> , and thus may alter cb activity by modifying coilin, a known phosphoprotein  <cit> .

 the biogenesis of most spliceosomal small nuclear ribonucleoproteins is complicated and requires both cytoplasmic and nuclear maturation steps. for example, the spliceosomal small nuclear rnas of ul, u u and u snrnps are synthesized by rna polymerase ii and may traffic through specific subnuclear domains before being exported to the cytoplasm. in the cytoplasm, a septet of sm proteins binds the sm motif of the snrna under the control of the survival of motor neurons protein complex. mutations in the smn protein cause the neurodegenerative disorder spinal muscular atrophy. after the sm core has been assembled onto the snrna, the snrna is subject to further processing, followed by import back into the nucleus; again with the help of the smn complex. upon nuclear re-entry, newly assembled ul, u u and u snrnps first localize to a subnuclear domain known as the cajal body. in the cajal body, the snrna component of the snrnp is subjected to pseudouridine-base and 2'-0-methyl sugar-modifications that are guided by small cb-specific rnas. these modifications are crucial for proper pre-mrna splicing in vivo. after their modification in the cb, snrnps localize to speckles, where they are stored, or perichromatin fibrils, where splicing occurs concurrently with transcription. unlike ul, u u and u snrnas, maturation of the rna polymerase iii-transcribed u snrna does not include a cytoplasmic phase and may take place in the nucleolus and the cb. the u snrnp, which is required for histone pre-mrna 3'-end processing, is assembled in manner similar to that observed for ul, u u and u snrnps, including a cytoplasmic phase. however, u snrna has a noncanonical sm binding site and thus recruits a different type of sm core compared to that which binds ul, u u and u snrna. like the spliceosomal snrnps, the u snrnp is enriched within cbs. cbs have been shown to move in an atp dependent manner as well as associate with various gene loci, including snrna and histone gene clusters. consequently, cbs may provide a platform upon which a feedback regulatory mechanism for snrnp and histone biogenesis takes place. the mechanisms by which snrnps are targeted to and released from the cb are unknown. one possibility is that a factor within the cb interacts with nascent snrnps and facilitates their modification. another factor may displace snrnps from the cb, allowing for their subsequent localization in speckles and perichromatin fibrils. the cb marker protein coilin may play a role in the targeting of snrnps to cbs. removal of coilin in xenopus by immunodepletion decreases snrnp levels in the amphibian equivalent of the cb. characterization of coilin knockout mice has revealed that they are viable on an outbred mouse strain, but have significant viability and fertility defects on inbred strains. cell lines derived from coilin knockout mice lack canonical cbs in which snrnps are enriched. however, add-back experiments demonstrate that typical cbs, containing snrnps, can be reformed upon the addition of coilin. furthermore, coilin can interact directly with several sm proteins of snrnps. taken together, these data indicate that, while not an essential protein, coilin is important for proper cb formation. functional cbs may allow for the efficient coordination of the nuclear steps of snrnp biogenesis. another protein that requires coilin for its localization to cbs is smn. coilin directly interacts with smn via several arginine/glycine dipeptide repeats in coilin. the arginines within this rg box motif are symmetrically dimethylated, resulting in an increased affinity for smn. in most cell lines and tissues, smn localizes to the cytoplasm and cbs. however, smn in some cell lines and fetal tissue localizes to discrete nuclear structures termed "gems". the presence of gems correlates with a decrease in coilin methylation. although the nuclear role of smn is not well understood, it is possible that smn escorts nascent snrnps to the cb. interestingly, coilin can compete with sm proteins for binding sites on smn, indicating that locally high concentrations of coilin within the cb may serve to displace snrnps from the smn complex. the interplay between smn, snrnps and coilin may therefore regulate snrnp accumulation within the cb. we set out to determine if we could identify factors that control the departure of nascent snrnps from the cb. in this report, we demonstrate that an isoform of the human mitochondrial tim tim50a, localizes to speckles and interacts with snrnps, smn and coilin. human tim which has phosphatase activity, is a component of the mitochondrial translocator and regulates mitochondrial integrity and cell death. the protein sequence of tim and tim50a are identical, with the important exception that tim50a contains an alternative translational start sequence that adds aa to its n-terminus relative to tim competition binding experiments show that smb' does not compete with smn for tim50a binding sites, but smb' does reduce coilin interaction with tim50a. furthermore, smn and coilin compete for tim50a binding sites and tim50a forms a more efficient complex with snrnps in vivo compared to tim based on these results, we propose that tim50a is involved in the regulation of snrnp biogenesis and possibly the activity of nuclear smn.  isolation of tim50a given the central role of coilin in the formation and composition of cbs, especially in its ability to recruit the smn complex and snrnps, we wanted to identify proteins that interact with coilin and possibly regulate smn and snrnp localization in the cb. towards this end, we conducted a yeast two-hybrid screen with coilin as bait. a human brain cdna library was chosen for this screen because we are interested in assessing cb protein dynamics in neuronal tissue, which is the cell type primarily affected in spinal muscular atrophy. the c-terminal aa of coilin, which interacts with smn and sm proteins, was the bait for the screen. consequently, other proteins that bind c may regulate the interplay between coilin/smn and coilin/sm and thus play a role in cb dynamics. several preys were recovered from the screen, one of which was termed cap sequencing of this prey revealed that it contained the partial coding sequence for a protein known as tim since tim localizes to the mitochondria and cytoplasm, the relevance of cap interaction with coilin, a nuclear protein, was not initially clear. however, upon conducting est database searches, we found that there are two possible translational starts for the timm gene product. the majority of ests terminate slightly upstream of the published translational start sequence of tim, suggesting that tim is the major product of the timm gene. however, three ests, all from cancer cells lines, demonstrate that an alternative translational start sequence can be utilized which would add aa to the n-terminus of the tim protein. we have named this protein tim50a. importantly, a putative nuclear localization signal is found within this region, indicating that tim50a, unlike tim may reside in the nucleus. to verify the existence of tim50a in other cell lines and to obtain a general idea as to the abundance of this transcript relative to the tim isoform, we performed nested pcr with primers specific to tim50a on cdnas from four additional cancer cell lines: a- nih: ovcar- mda-mb- and hct- another primer set was used in a standard pcr reaction to amplify a region common to both tim50a and tim. as shown in figure 1c, a standard pcr reaction using primers that can bind both tim50a and tim generate a product from all of the cell lines tested. in contrast, standard pcr reactions using tim50a-specific primers fail to yield a product from cell line cdnas, but successfully generate a product using a positive control plasmid template. however, nested pcr reveals that two of the four cell lines, a- and nih: ovcar- contain the tim50a transcript, as monitored by sequencing of the product. the lower band in the nih: ovcar- reaction is from an unrelated transcript. therefore, while tim50a mrna is not nearly as abundant as the tim message, this species is present in at least four different cell lines. cellular localization of tim50a to test if tim50a is a nuclear protein, we obtained an est encoding the additional residues and cloned the entire tim50a coding sequence into a gfp expression vector. we also made a gfp fusion to the truncated tim protein isolated from the two-hybrid screen as well as gfp fusions to various mutants of tim50a. the localization of these proteins, and gfp fused to the mitochondrial tim isoform, was monitored after transient transfection into hela cells. in contrast to tim gfp-tim50a is exclusively nuclear with speckle-like accumulations. to confirm that the nuclear accumulations are indeed speckles, in which snrnps are stored, we stained the cells with antibodies against sm proteins. there is overlap between tim50a and snrnps in speckles, as shown by the yellow-orange signal in the merged image. we do not detect tim50a in cajal bodies, indicating that the association of tim50a with cbs or coilin is weak or transient. alternatively, coilin may associate with tim50a in the nucleoplasm. indeed, while coilin is highly enriched in the cb, the majority of the protein is diffusely localized throughout the nucleoplasm. we observed identical localization patterns for tim50a upon fusion to a smaller myc-tag, suggesting that the gfp-tag does not affect localization. the diffuse cytoplasmic and nuclear localization of gfp-cap and gfp-tim50a demonstrate that the region containing the putative nls is necessary for nuclear localization. tim50a mutants that retain the putative nls display nuclear accumulation. for example, an internal deletion of tim50a is found in a speckled nuclear pattern while the first aa is nuclear and nucleolar. tim50a localization to speckles is therefore contingent upon the nls and residues found between aa and actinomycin d redistribution of tim50a the transcription inhibitor actinomycin d can be used to partially characterize the interaction of proteins in the nucleus. this is because actinomycin d inhibits transcription and causes the reorganization of splicing factors and cajal bodies. in particular, speckles enlarge and cbs are relocalized to the periphery of the nucleolus in the presence of actinomycin d. if tim50a were an integral part of speckles, then we would suspect that this protein would be found in enlarged speckles when cells are treated with actinomycin d. however, in the presence of actinomycin d, tim50a no longer localizes with snrnps in speckles but is instead found in discrete structures. this is evident in the merged image, which shows separate green and red signals with little overlap. in contrast, non-treated cells display overlap between tim50a and snrnps, resulting in a yellow-orange signal in the merged image. we tested whether tim50a in actinomycin d treated cells localized to paraspeckles, another subnuclear domain of unknown function. no colocalization of tim50a with the paraspeckle marker protein, psp was observed. taken together, the localization data indicate that tim50a is likely to have a different function compared to tim possibly in the regulation of cb activity or snrnp biogenesis. however tim50a is not an integral component of speckles given the separate localization pattern of tim50a and snrnps in actinomycin d treated cells. direct interaction of tim50a with coilin, smb' and smn the two-hybrid screen suggests that tim isoforms may interact with coilin. to prove this, we conducted gst-pulldown assays using bacterially purified t7-tagged coilin and tim50a fused to gst. coilin is not recovered over beads fused to gst alone, but is recovered over gst-tim50a beads. we next tested if tim50a would interact with the c-terminal aa of coilin, which is this same fragment of coilin used to isolate cap in the two-hybrid screen. in agreement with the two-hybrid results, soluble tim50a is recovered over gst-c beads, but not by gst alone. since tim50a localizes to speckles, we were interested in assessing if tim50a interacts with sm proteins of snrnps. using smb' as a representative of sm proteins, we conducted gst-pulldown experiments with a variety of gst-tim50a proteins. consistent with the localization data, smb' directly interacts with gst-tim50a. smb' also associates with two mutants of tim50a, cap and tim50sac. based on the binding results, we suspect that the interaction domain for smb' on tim50a lies somewhere between residues and of tim50a, although we cannot exclude the role of the residues at the extreme c-terminus of tim50a. given that tim50a localizes to speckles and directly interacts with coilin and smb', we next tested if tim50a could interact with another protein crucial for snrnp formation, smn. as shown in figure 6d, smn binds to the gst-tim50a proteins but not to gst alone. we also examined if the isoform known to localize in mitochondria, tim also interacts with the coilin fragment c as found for tim50a. purified full-length tim does indeed interact specifically with the coilin fragment as shown in figure 6e. from these in vitro binding studies, we conclude that coilin, smb' and smn can interact with both tim50a and tim isoforms, but the interaction of these proteins in vivo is likely regulated by their subcellular localization. smb' and smn compete with coilin for tim50a binding sites the ability of tim50a to bind coilin, smb' and smn raises the interesting possibility that this protein may regulate the dynamics of cb proteins. to test this hypothesis, we conducted competition gst-pulldown experiments using gst-tim50asac. this protein was used instead of gst-tim50a because it is more easily purified compared to gst-tim50a and has similar binding capabilities. for the first experiment, we tested if smn and smb' vie for the same binding sites on tim50a. this was accomplished by incubating a fixed amount of gst-tim50asac with a constant amount of smn and, in separate reactions, an increasing amount of smb'. despite binding a large amount of smb', the level of smn recovered by the gst-tim50asac beads does not change. we conclude that tim50a has separate binding sites for smn and smb' and that one protein does not preclude the other from binding. we then tested if coilin and smb' compete for binding sites on tim50a. for this experiment, we incubated a fixed amount of gst-tim50asac with a constant amount of a coilin fragment and, in separate reactions, an increasing amount of smb'. as shown in figure 7b, increasing the level of smb' decreases the recovery of the coilin fragment by gst-tim50asac. thus coilin and smb' compete for binding sites on tim50a. finally we monitored if coilin and smn compete for the same binding sites on tim50a. this experiment was conducted with a fixed amount of gst-tim50asac and a constant amount of smn with increasing levels of the coilin fragment. the binding of the respective proteins to the tim50a substrate in the absence of competitor is shown in lane for the coilin fragment and lane for smn. in reactions with both soluble proteins, as the level of coilin fragment is increased, the amount of smn recovered by the tim50a beads is reduced. these data are consistent with a competition for tim50a binding sites by smn and coilin. given its protein interaction potential, we hypothesize that tim50a may affect cb function by interacting with proteins that localize to this domain. one possible scenario is that tim50a displaces snrnps from the cb and facilitates their translocation to speckles. tim50a may also disrupt the interaction between coilin and smn, allowing for smn to leave the cb. tim50a interacts with smn and snrnps in vivo to corroborate the direct interaction data, we tested if tim50a could associate with smn in cells. for these experiments, we transfected hela cells with empty gfp vector, gfp-tagged full-length tim50a or gfp fused to the n-terminal aa of tim50a. after incubation, lysates were made and subjected to immunoprecipitation with anti-gfp antibodies, followed by western blotting with anti-smn antibodies. as shown in figure 8a, no smn is co-immunoprecipitated with gfp alone but smn is recovered by gfp-tim50a. only a faint smn signal is observed in the gfp-tim50a reaction, suggesting that this fragment does not contribute significantly to the ability of smn to bind tim50a. reprobing of this same blot with anti-gfp demonstrates that while a large amount of gfp and gfp-tim50a were immunoprecipitated, little smn was recovered. in contrast, the amount of immunoprecipitated gfp-tim50a is less than that obtained for gfp and gfp-tim50a, yet more smn was recovered. thus tim50a can form a complex with smn in cells. given that tim50a localizes to speckles, in which snrnps are stored, and directly interacts with a subunit of the sm core, it is likely that tim50a will interact with intact snrnps. however, the in vitro binding data indicate that the mitochondrial isoform, tim also has the potential to interact with snrnps, which are known to have a cytoplasmic phase. to test this, we conducted a co-immunoprecipitation assay on hela cells transfected with empty gfp vector or gfp fused to tim50a or tim lysates were generated and subject to incubation with the anti-sm antibody y after sds-page and western transfer, the blot was probed with anti-gfp antibodies to monitor the association of gfp-tim50a or gfp-tim with snrnps. as shown in figure 8b, a signal corresponding to gfp-tim50a is present in the y immunoprecipitation reaction. in contrast, less tim is co-immunoprecipitated by y compared to tim50a. a gfp-alone signal is not observed in the y reaction, demonstrating the specificity of the reaction. therefore, in vivo, tim50a appears to be more efficient in snrnp interaction than tim although we have not excluded the possibility that the gfp-tag may adversely affect tim interactions. to address this point, tim50a and tim proteins were fused to smaller myc-tags, expressed in hela cells and tested for interaction with an immobilized sm protein, smb'. both proteins bind gst-smb', indicating that the gfp-tag may indeed contribute to the reduced interaction of tim with snrnps. alternatively, the faint tim signal observed in the y co-immunoprecipitation reaction may be an artifact due to the overexpression of gfp-tim and subsequent nuclear localization, as shown in figure in support of this thought, immunofluorescence analysis of endogenous tim demonstrates that this protein is predominantly cytoplasmic with little nuclear accumulation. given that tim50a is a nuclear protein, we speculate that tim50a interactions may regulate the nuclear activity of smn and trafficking of snrnps, possibly by altering the dynamics of the association between smn, coilin and snrnps. additionally, it is plausible that cytoplasmic smn can interact with tim before mitochondrial import.  the cellular localization and protein interaction characteristics of tim50a suggest that this isoform of tim may play a role in snrnp biogenesis. based on the abundance of ests, it is likely that tim not tim50a, is the major product of the timm gene. however, the presence of tim50a ests from cancer cell lines suggests that this protein may be induced in cellular transformation. it is known that cancer cells contain cbs, while some normal cells and tissues do not. for example, normal adult lung tissue does not contain cbs, but lung cancer cells do. since cbs play a role in snrnp biogenesis, it is likely that the increase of cbs in transformed cells reflects on the heightened demand in splicing requirements for upregulated genes that participate in the establishment and/or maintenance of the altered cellular physiology. the induction of cbs may also facilitate the production of telomerase, the rna component of which localizes to cbs. in addition to cancer cells neuronal cells contain robust cbs, possibly because high levels of snrnps are necessary to process the abundance of messages subject to alternative splicing in neuronal tissue. therefore, tim50a expression may be limited to tissue with high demands in snrnp metabolism. we are currently developing an antibody to the unique n-terminus of tim50a in order to explore this possibility. additionally, experiments are underway to verify the role of tim50a in snrnp biogenesis. in particular, we are interesting in determining if tim50a is involved in the liberation of snrnps from the cb after they have been modified. the functional consequence of the interaction between tim50a and smn is also being investigated. finally we note that tim50a contains the phosphatase domain shown to be functional in the tim isoform, and thus may alter cb activity by modifying coilin, a known phosphoprotein. 
the cajal body is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins, which are vital for pre-mrna splicing. the tim50a transcript can be detected in some cancer cell lines and normal brain tissue. newly imported sm-class snrnps traffic through cbs, where the snrna component of the snrnp is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. tim50a may play a role in snrnp biogenesis given its cellular localization and protein interaction characteristics. in addition to coilin, tim50a interacts with snrnps and smn. the marker protein for cbs, coilin, may play a role in snrnp biogenesis given that it can interact with snrnps and smn, the protein mutated in spinal muscular atrophy. competition binding experiments demonstrate that coilin competes with sm proteins of snrnps and smn for binding sites on tim50a. loss of coilin function in mice leads to significant viability and fertility problems and altered cb formation.
 the cajal body is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins, which are vital for pre-mrna splicing. newly imported sm-class snrnps traffic through cbs, where the snrna component of the snrnp is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. it is not known how nascent snrnps localize to the cb and are released from this structure after modification. the marker protein for cbs, coilin, may play a role in snrnp biogenesis given that it can interact with snrnps and smn, the protein mutated in spinal muscular atrophy. loss of coilin function in mice leads to significant viability and fertility problems and altered cb formation.  in this report, we identify a minor isoform of the mitochondrial tim tim50a, as a coilin interacting protein. the tim50a transcript can be detected in some cancer cell lines and normal brain tissue. the tim50a protein differs only from tim in that it contains an additional aa n-terminal to the translation start of tim importantly, a putative nuclear localization signal is found within these residues. in contrast to tim which localizes to the cytoplasm and mitochondria, tim50a is strictly nuclear and is enriched in speckles with snrnps. in addition to coilin, tim50a interacts with snrnps and smn. competition binding experiments demonstrate that coilin competes with sm proteins of snrnps and smn for binding sites on tim50a.  tim50a may play a role in snrnp biogenesis given its cellular localization and protein interaction characteristics. we hypothesize that tim50a takes part in the release of snrnps and smn from the cb. 