BACKGROUND
the celf /bruno-like protein family plays important roles in the regulation of alternative splicing and translation  <cit> .
in mammals, the celf/bruno-like family includes six members and is classified into two subgroups based on overall sequence similarity.
one group is composed of cugbp <dig> and cugbp <dig>  which share 76% amino acid sequence identity  <cit> .
the other group contains brunol <dig> , brunol <dig> , brunol <dig> , and celf <dig>  which share 62-66% a.a. sequence identity with each other and 44% sequence identity with cugbp <dig>  <cit> .
celf proteins have two consecutive rna recognition motifs   in the n-terminal region and another rrm  in the c-terminal region.
rrm <dig> and rrm <dig> are separated by a linker region that consists of 160- <dig> amino acids.
celf family members are expressed in multiple tissues with a distinct tissue distribution pattern.
cugbp <dig> is expressed in almost all tissues, brunol <dig> and brunol <dig> are restricted to the brain, and cugbp <dig> is abundant in the heart, skeletal muscle, and brain  <cit> .
the celf family protein cugbp <dig> was originally identified as an rna-binding protein for cug triplet repeats observed in the 3' utr of the dystrophy myotonica protein kinase  mrna  <cit> .
increased copies of cug triplet repeats from 5- <dig> to 50- <dig>  cause myotonic dystrophy type i. cugbp <dig> regulates the alternative splicing of exon  <dig> of cardiac troponin t  via the cug repeats of muscle-specific enhancer elements  in its pre-mrnas  <cit> .
cugbp <dig> is presumably involved in the incidence of dm because the splicing of ctnt was disrupted in dm striated muscle.
in addition to its role in the regulation of ctnt exon  <dig> splicing, cugbp <dig> is thought to be involved in controlling the alternative splicing of amyloid precursor protein   <cit> , muscle-specific chloride channel  <cit> , α-actinin   <cit> , and the insulin receptor   <cit> .
cugbp <dig> , a paralogous protein of cugbp <dig>  is also known as an alternative splicing regulator.
similar to cugbp <dig>  cugbp <dig> activates the inclusion of exon  <dig> in human ctnt mrna via binding to mse  <cit> .
cugbp <dig> induces smooth muscle-specific exon inclusion via binding to uridine purine repeat elements  in actn <dig>  <cit> .
although cugbp <dig> is known as a splicing activator for the n-methyl-d-aspartate receptor  <dig>  exon  <dig>  cugbp <dig> represses nmda r <dig> exon  <dig> inclusion  <cit> .
in addition, cugbp <dig> was reported to repress the inclusion of the ir exon  <dig>  <cit> .
these studies showed that cugbp <dig> has positive and negative regulatory roles in alternative splicing.
bruno, another member of this protein family, is a translational repressor involved in germ cell formation in drosophila  <cit> .
the bruno protein binds to the bre  of the 3' utr of oskar mrna and represses its translation in the oocyte.
the oskar protein is responsible for germ cell formation in the cytoplasm of the posterior pole of the oocyte.
in vertebrates, eden-bp , an orthologous protein of cugbp <dig> in xenopus, has been reported to control the translational regulation activity of specific maternal mrnas via the eden sequence  <cit> .
in addition, not only bruno-like mrna but also its protein, which is an orthologous protein of cugbp <dig> in zebrafish, localized to the germplasm at the end of the cleavage furrow  <cit> .
these results suggest that cugbp <dig> is involved in translational control and germ cell formation.
the binding sequences of cugbp <dig> and cugbp <dig> can be classified into two groups.
one group is represented by cug triplet repeats of ctnt, mt-pk, dmpk and c/ebpβ  <cit> , where the binding sequence essentially contains cug repeats.
another group is represented by the bre of oskar, eden of eg <dig> and ure of actn <dig>  <cit> , and the binding sequence essentially contains uridine and guanine -repeats.
although there are three rrms responsible for rna-binding in cugbps, previous studies concluded that the rrm <dig> of cugbps binds to the ug-repeat in a sequence-specific manner  <cit> .
contradictory results showed that consecutive rrm1-2s bind to rna in a sequence-specific manner.
in addition to rrms, the amino acids surrounding the rrms were reported to affect rna-binding and splicing activities.
indeed, rrm1- <dig> plus  <dig> residues of the adjacent downstream linker and rrm <dig> plus the last  <dig> amino acids of the adjacent upstream linker activated the mse-dependent exon inclusion of ctnt  <cit> .
most of the mammalian genes, including cugbp <dig>  are transcribed as alternatively spliced variants.
among the alternative exons of cugbp <dig>  the present study focused on the skipping of exon  <dig>  which encodes the first half of rrm <dig>  generally, an rrm is composed of two α-helixes and four β-sheets  formed by almost  <dig> a.a.  <cit> .
the well-conserved rnp- <dig> and rnp- <dig> correspond to the third β-sheet  and first β-sheet , respectively.
rnp- <dig> is important for the intercalation of target rna in a sequence-specific manner  <cit> .
because exon  <dig> of the cugbp <dig> gene encodes the first half of rrm <dig>  a skipping transcript produces cugbp <dig> truncated in parts of rrm <dig> .
similar to cugbp <dig> r3δ, there are many proteins that encode unusual or partially truncated rrm domains in the database .
u2af <dig> has an unusual rrm called the u <dig> homology motif  and its interaction with u2af <dig> strengthens rna binding  <cit> .
moreover, many proteins that contain partially deleted rrms appear to be generated by alternative splicing.
however, it is unclear how these proteins with partially truncated rrms caused by alternative splicing function in biological processes.
the list shows human proteins possessing an rrm with a deleted part selected from human refseq proteins.
in  <dig> human among  <dig> human rrm-type proteins,  <dig> human refseq proteins  showed a severely disrupted rrm domain.
when two rrm domains have a short linker region , these rrms were considered as tandem rrms.
when an alternatively spliced isoform restored the complete rrm, the name of the isoform was listed.
the parentheses show rrms that were removed form the alternatively spliced isoforms.
the present study examined the expression patterns of cugbp <dig> mrna and its isoform, r3δ, in p <dig> cells during neural differentiation and in different tissues.
cugbp <dig> was highly expressed in neural cells and in the adult brain compared with r3δ, which was the major product in the kidneys, liver and undifferentiated p <dig> cells.
transient transfection experiments showed similar activities of cugbp <dig> and r3δ, and both proteins promoted the use of the sm  exon instead of the nm  exon of the actn <dig> minigene.
on the other hand, cugbp <dig> and r3δ had opposite effects on alternative splicing of exon  <dig> of ir; cugbp <dig> repressed ir exon  <dig> inclusion, whereas r3δ did not and even slightly increased inclusion.
this result suggests that the alternatively spliced isoform, r3δ, has a different function from that of cugbp <dig>  in addition, the results of molecular dynamics  and nmr showed that the structure of the rrm domain differs significantly from that in the r3δ isoform, resulting in the disruption of its binding activity.
RESULTS
alternative splicing of cugbp2
a search of the ucsc genome browser, blat, suggested that the r3δ isoform is the product of skipping of exon  <dig> in 25% of cugbp <dig> transcripts.
exon <dig> is  <dig> nt and the skipping transcript does not generate a new premature termination codon.
to assess in which organ alternative splicing of rrm <dig> in cugbp <dig> takes place, rt-pcr was performed in adult mouse tissues.
the products of exon  <dig> inclusion  and exon  <dig> skipping , which encode a complete and partial rrm <dig>  respectively, were detected .
cugbp <dig> mrna was highly expressed in the brain, where the main product was the exon  <dig> inclusion transcript encoding a complete rrm <dig> .
the total amount of cugbp <dig> transcripts in the kidney or liver was low compared to that in brain, but the percentage of the r3δ isoform was relatively high .
these results suggest that the r3δ isoform is one of major products when their gene expression of cugbp <dig> is low, and the cugbp <dig> isoform, but not r3δ, is expressed as the major product when their gene expression is high.
to further investigate the alternative splicing pattern of cugbp <dig>  rt-pcr was performed in p <dig> cells during neural differentiation.
increased levels of the exon  <dig> inclusion product were detected during neural differentiation even though the exon  <dig> skipping product was not essentially changed .
the r3δ percentage on day  <dig>  was  <dig> %, which was the lowest during p <dig> neural differentiation .
the relatively low expression of r3δ with respect to alternative splicing patterns was also found in tissues of the adult mouse.
western blot analysis of cugbp <dig> proteins showed that the full length isoform was the main protein in p <dig> cells and that its level increased at the neural stage.
by contrast, the level of r3δ decreased at the neural stage, although it was observed in undifferentiated p <dig> cells .
expression analysis of cugbp <dig> target genes, actn <dig> and insulin receptor
cugbp <dig> is a regulator of the alternative splicing of several transcripts, including actn <dig>  <cit>  and ir  <cit> .
the alternative splicing of actn <dig> and ir was therefore analyzed in adult mouse tissues and p <dig> cells.
the actn <dig> gene has mutually exclusive exons, namely the smooth muscle  exon and non-muscle  exon .
the sm exon., as a percentage of nm and sm exons, was higher in neural differentiated p <dig> cells  than in undifferentiated p <dig> cells .
in the brain,  <dig> % of actn <dig> transcripts contained the sm exon, while the nm exon was predominant in the kidney  and liver .
cugbp <dig> has been suggested to promote the inclusion of the sm exon in prior work  <cit> , suggesting that the elevated expression of cugbp <dig> and inclusion of the actn <dig> sm exon may occur in the same cells and tissues.
on the other hand, the ir gene consists of  <dig> exons and generates  <dig> isoforms, ir-a and ir-b, which are characterized by the skipping and inclusion of exon  <dig>  respectively   <cit> .
rt-pcr analysis of the alternative splicing of the ir gene in tissues and p <dig> cells identified the two isoforms, ir-a and ir-b, in p <dig> cells during neural differentiation .
although the expression of the ir gene increased during the glial cell stage, the ira/ir-b ratio did not change significantly during cell differentiation .
while the exon  <dig> skipping variant ir-a was expressed in the brain , the exon  <dig> inclusion form ir-b was mainly expressed in the kidney  and liver .
because cugbp <dig> is known to repress the inclusion of ir exon  <dig>  <cit> , elevated expression of cugbp <dig> and the ir-a variant may occur in the same tissues, but is not observed in p <dig> cells.
in addition to the expression of cugbp <dig>  the ratio of r3δ to cugbp <dig> was lower in the brain than in the kidney and liver.
the function of the r3δ isoform was therefore examined in detail as described in the next section.
cugbp <dig> r3δ induces smooth muscle type splicing of actn1
as described previously, cugbp <dig> promotes the inclusion of the mutually exclusive sm exon instead of the nm exon in actn <dig>  <cit> .
the specific function of the cugbp <dig> isoform r3δ was examined by transient transfection using the mouse actn <dig> minigene .
transfection of cos <dig> cells with the actn <dig> minigene alone resulted in the detection of a transcript including the nm exon as a major product and another transcript with the sm exon as a minor product .
control cos <dig> cells lacking the minigene did not show these transcripts, confirming that the two transcripts containing the nm or sm exon were products of the transfected minigene.
co-transfection with a myc-tagged cugbp <dig> expression vector resulted in a decrease in the nm exon product concomitant with an increase in the sm exon product .
use of the etr- <dig> expression vector as a control showed that etr- <dig> promoted the use of the nm exon.
these results are consistent with previous reports  <cit> .
to assess the function of the alternatively spliced isoform of cugbp <dig> r3δ, transient transfection experiments were performed using the actn <dig> minigene with the r3δ instead of the cugbp <dig> expression vector.
r3δ induced the production of the mutually exclusive sm exon similar to cugbp <dig> , suggesting that the function of r3δ in actn <dig> splicing is similar to that of cugbp <dig> and that part of rrm <dig>  which is encoded in r3δ, may be dispensable for the regulation of the splicing of actn <dig>  as shown above, our results identified the brain as the location of sm exon inclusion and the kidney and liver as characteristic of nm exon inclusion.
in parallel to sm exon inclusion, the expression level of cugbp <dig> was high in the brain and low in the kidney and liver, suggesting that the upregulation of cugbp <dig> in the brain contributes to the induction of sm exon inclusion.
different effects of cugbp <dig> isoforms on the insulin receptor
it was reported that cugbp <dig> induces the skipping of exon  <dig> in ir  <cit> .
the induction of exon  <dig> skipping by cugbp <dig> was assessed using the ir minigene in transient transfection experiments .
transfection of cos <dig> or hela cells with the ir minigene alone resulted in the production of similar amounts of exon  <dig> inclusion and skipping products .
exon  <dig> inclusion and skipping products were not detected in cos <dig> or hela cells without the minigene, confirming that they were transcribed from the transfected minigene.
co-transfection of the cugbp <dig> expression vector and the ir minigene caused an increase in the exon  <dig> skipping product and a decrease in the exon  <dig> inclusion product .
co-transfection of etr- <dig> as a control did not cause significant changes compared with the transfection of the minigene alone .
these results confirmed that cugbp <dig> induces the skipping of exon  <dig>  as previously reported.
this effect was clearer in hela cells than in cos <dig> cells .
to examine potential changes in the function of the alternatively spliced isoform, r3δ, the ir minigene was co-transfected with the r3δ expression vector, which caused a decrease in exon  <dig> skipping and an increase in exon  <dig> inclusion in comparison to co-transfection with cugbp <dig> or the mock control .
in a previous report, artificially truncated cugbp <dig> proteins with deleted n-terminal regions or deleted c-terminal regions were unable to engage in efficient exon  <dig> skipping  <cit> .
in the present study, r3δ promoted the exon  <dig> inclusion, which is in contrast to the activity to full-length cugbp <dig>  which promotes exon  <dig> exclusion of the ir gene.
these results also indicate that part of rrm <dig>  which is encoded by the alternative exon  of cugbp <dig>  is not only essential for the skipping of ir exon  <dig>  but that disruption of this part of rrm <dig> may result in a change in the splicing regulated by cugbp <dig>  among celf family proteins, an isoform resembling r3δ can be predicted in celf <dig> .
however, a generalized rule for rrm3s cannot be established because exon  <dig> skipping can be activated by a subgroup of proteins that includes cugbp <dig> and  <dig>  but not by another subgroup that includes etr- <dig>  or celf <dig>   <cit> .
our results suggest that the disruption of part of rrm <dig> in cugbp <dig> generates a new activity in the regulation of the splicing of the ir gene.
because the r3δ isoform has a similar activity to cugbp <dig> in the regulation of splicing of actn <dig>  it cannot be considered simply as a dominant negative isoform of cugbp <dig>  the product of exon  <dig> skipping was detected in the brain and the product of exon  <dig> inclusion was detected in the kidney and liver, suggesting that high cugbp <dig> expression in the brain contributes to exon  <dig> skipping.
a high ratio of r3δ to cugbp <dig> in the kidney and liver may repress exon  <dig> skipping due to the specific function of the r3δ isoform.
structural analyses of the third rrm of the cugbp <dig> r3δ isoform
a total of  <dig> human refseq proteins with rrm domain were found in the blast.
an rrm domain is usually  <dig> ~  <dig> a.a. in length, and slightly smaller rrm domains , which appear to lack rnp- <dig> in the n-terminus or the dimerization module in the c-terminal region, are frequently observed in conserved domain searches of blast.
rrm <dig> of cugbp <dig> consists of  <dig> a.a., and  <dig> a.a. of this region are disrupted in the r3δ isoform.
disruption of almost half of rrm did not prevent recognition of the remaining sequence in the conserved domain search.
a total of  <dig> human refseq proteins  with severely disrupted rrm domains are listed in table  <dig>  some of the domains shown in table  <dig> such as eif3b, eif4b, la , u2af <dig>  and u2af <dig> , have been reported to form unusual rrm structures, although the amino acid sequences were not sufficient to predict specific rrm structures  <cit> .
there-dimensional structures of the rrm <dig> isoforms were examined by md simulation and nmr spectroscopy.
the structural analysis of the rrm <dig> of cugbp <dig> has been reported previously  <cit> .
several unaligned residues were found between the rrm3s of cugbp <dig>  and  <dig>  the results of homology modeling showed that the third domain of cugbp <dig> formed an rrm structure .
no significant difference could be observed between the md simulations of rrm <dig> of cugbp <dig> and rrm <dig> of cugbp <dig>  .
further, cugbp <dig> rrm <dig> maintained an rrm structure for  <dig> ns in the md simulation .
the 1h-15n hsqc spectrum of the rrm <dig> of cugbp <dig> in the absence of rna showed that, unlike rrm <dig> of cugbp <dig>  the resonances of the n-terminal residues in the linker domain were concentrated in the center of the spectrum, suggesting a random coil structure.
this result was probably due to the inclusion of a long n-terminal sequence in the present constructs, which forms a flexible linker region connecting rrm <dig> with rrm <dig> .
protein threading and comparative modeling of the r3δ isoform were performed in the pdbj.
the rrm <dig> of human cugbp <dig> was chosen as the ideal template in the search and it was predicted that the r3δ isoform with part of the linker sequence would not form an rrm structure, especially as the first α-helix  was missing .
we used md simulations to investigate whether the addition of linker residues to the rrm domain could restore its structure.
however, even in these conditions, the first α-helix cannot be formed even after  <dig> ns .
moreover, resonances corresponding to the r3δ isoform were detected in the central region of the nmr spectrum, suggesting the presence of greater alterations in the structure of r3δ than those shown by md simulation  and indicating that the rrm structure cannot be restored in the r3δ isoform.
in addition, the interaction between cugbp <dig> or the r3δ isoform and rna molecules was examined.
the addition of rna caused the disappearance of many resonances and the appearance of new resonances in cugbp <dig> .
the affected residues, which are highlighted with an open box in figure 5b, were consistent with the corresponding region of cugbp <dig>  <cit>  and indicate that cugbp <dig> binds to  <dig> in a similar manner as cugbp <dig>  no spectral changes were detected in the r3δ isoform with the addition of rna , indicating that the r3δ isoform does not bind to  <dig>
although the rna-binding of rrm1- <dig> of cugbps is still unclear, rrm <dig> is known to be responsible for rna-binding to ug-repeats  <cit> .
md simulation suggested that the r3δ isoform could not form an rrm domain and the nmr spectrum showed that it did not bind to the ug-repeat.
these results lead to the speculation that the disruption of the first half of rrm <dig> by exon  <dig> skipping causes a defect in rna-binding.
however, the r3δ isoform may interact with actn <dig> pre-mrna based on the fact that this isoform still has two rrms and two different cis-elements were reported in the alternative splicing of actn <dig>  <cit> .
a cugbp <dig> interacting cis-element was determined in the intronic region of ir and shown to affect the alternative splicing of ir  <cit> .
because this sequence is not a typical ug-repeat, perhaps the rrm <dig> may not be responsible for the primary interaction with the ir pre-mrna.
therefore, other, currently unknown, proteins might interact with the rrm <dig> domain and it might be the interactions of these proteins rather than rrm <dig> rna-binding activity that is abrogated by rrm <dig> alternative splicing.
CONCLUSIONS
the results of the present study show that while cugbp <dig> and its r3δ isoform similarly promoted the use of the sm exon instead of the nm exon in the alternative splicing of the actn <dig> minigene, they did not similarly promote the use of exon  <dig> in the alternative splicing of the ir minigene; cugbp <dig> promoted skipping of exon  <dig>  whereas r3δ did not.
in addition, the results of md simulation and nmr suggested that the truncated rrm <dig> region in r3δ resulting from the alternative splicing of cugbp <dig> could neither form a new rrm domain nor bind to a ug-repeat.
thus, it is possible that other, currently unknown, cugbp2-interacting proteins and/or other splicing factors involved in splicing regulation might be important for rrm <dig> function.
our results also showed that the skipping of exon  <dig> of cugbp <dig> not only disrupted cugbp <dig> rna-binding activity but also altered its splicing regulator function.
a high ratio of r3δ to cugbp <dig> in the kidney and liver may affect the splicing of the ir gene and repress exon  <dig> skipping.

 the celf /bruno-like protein family plays important roles in the regulation of alternative splicing and translation. in mammals, the celf/bruno-like family includes six members and is classified into two subgroups based on overall sequence similarity. one group is composed of cugbp and cugbp which share 76% amino acid sequence identity. the other group contains brunol, brunol, brunol, and celf which share 62-66% a.a. sequence identity with each other and 44% sequence identity with cugbp. celf proteins have two consecutive rna recognition motifs in the n-terminal region and another rrm in the c-terminal region. rrm and rrm are separated by a linker region that consists of 160- amino acids. celf family members are expressed in multiple tissues with a distinct tissue distribution pattern. cugbp is expressed in almost all tissues, brunol and brunol are restricted to the brain, and cugbp is abundant in the heart, skeletal muscle, and brain. the celf family protein cugbp was originally identified as an rna-binding protein for cug triplet repeats observed in the 3' utr of the dystrophy myotonica protein kinase mrna. increased copies of cug triplet repeats from 5- to 50- cause myotonic dystrophy type i. cugbp regulates the alternative splicing of exon of cardiac troponin t via the cug repeats of muscle-specific enhancer elements in its pre-mrnas. cugbp is presumably involved in the incidence of dm because the splicing of ctnt was disrupted in dm striated muscle. in addition to its role in the regulation of ctnt exon splicing, cugbp is thought to be involved in controlling the alternative splicing of amyloid precursor protein, muscle-specific chloride channel, α-actinin, and the insulin receptor. cugbp, a paralogous protein of cugbp is also known as an alternative splicing regulator. similar to cugbp cugbp activates the inclusion of exon in human ctnt mrna via binding to mse. cugbp induces smooth muscle-specific exon inclusion via binding to uridine purine repeat elements in actn. although cugbp is known as a splicing activator for the n-methyl-d-aspartate receptor exon cugbp represses nmda r exon inclusion. in addition, cugbp was reported to repress the inclusion of the ir exon. these studies showed that cugbp has positive and negative regulatory roles in alternative splicing. bruno, another member of this protein family, is a translational repressor involved in germ cell formation in drosophila. the bruno protein binds to the bre of the 3' utr of oskar mrna and represses its translation in the oocyte. the oskar protein is responsible for germ cell formation in the cytoplasm of the posterior pole of the oocyte. in vertebrates, eden-bp, an orthologous protein of cugbp in xenopus, has been reported to control the translational regulation activity of specific maternal mrnas via the eden sequence. in addition, not only bruno-like mrna but also its protein, which is an orthologous protein of cugbp in zebrafish, localized to the germplasm at the end of the cleavage furrow. these results suggest that cugbp is involved in translational control and germ cell formation. the binding sequences of cugbp and cugbp can be classified into two groups. one group is represented by cug triplet repeats of ctnt, mt-pk, dmpk and c/ebpβ, where the binding sequence essentially contains cug repeats. another group is represented by the bre of oskar, eden of eg and ure of actn, and the binding sequence essentially contains uridine and guanine -repeats. although there are three rrms responsible for rna-binding in cugbps, previous studies concluded that the rrm of cugbps binds to the ug-repeat in a sequence-specific manner. contradictory results showed that consecutive rrm1-2s bind to rna in a sequence-specific manner. in addition to rrms, the amino acids surrounding the rrms were reported to affect rna-binding and splicing activities. indeed, rrm1- plus residues of the adjacent downstream linker and rrm plus the last amino acids of the adjacent upstream linker activated the mse-dependent exon inclusion of ctnt. most of the mammalian genes, including cugbp are transcribed as alternatively spliced variants. among the alternative exons of cugbp the present study focused on the skipping of exon which encodes the first half of rrm generally, an rrm is composed of two α-helixes and four β-sheets formed by almost a.a.. the well-conserved rnp- and rnp- correspond to the third β-sheet and first β-sheet, respectively. rnp- is important for the intercalation of target rna in a sequence-specific manner. because exon of the cugbp gene encodes the first half of rrm a skipping transcript produces cugbp truncated in parts of rrm. similar to cugbp r3δ, there are many proteins that encode unusual or partially truncated rrm domains in the database. u2af has an unusual rrm called the u homology motif and its interaction with u2af strengthens rna binding. moreover, many proteins that contain partially deleted rrms appear to be generated by alternative splicing. however, it is unclear how these proteins with partially truncated rrms caused by alternative splicing function in biological processes. the list shows human proteins possessing an rrm with a deleted part selected from human refseq proteins. in human among human rrm-type proteins, human refseq proteins showed a severely disrupted rrm domain. when two rrm domains have a short linker region, these rrms were considered as tandem rrms. when an alternatively spliced isoform restored the complete rrm, the name of the isoform was listed. the parentheses show rrms that were removed form the alternatively spliced isoforms. the present study examined the expression patterns of cugbp mrna and its isoform, r3δ, in p cells during neural differentiation and in different tissues. cugbp was highly expressed in neural cells and in the adult brain compared with r3δ, which was the major product in the kidneys, liver and undifferentiated p cells. transient transfection experiments showed similar activities of cugbp and r3δ, and both proteins promoted the use of the sm exon instead of the nm exon of the actn minigene. on the other hand, cugbp and r3δ had opposite effects on alternative splicing of exon of ir; cugbp repressed ir exon inclusion, whereas r3δ did not and even slightly increased inclusion. this result suggests that the alternatively spliced isoform, r3δ, has a different function from that of cugbp in addition, the results of molecular dynamics and nmr showed that the structure of the rrm domain differs significantly from that in the r3δ isoform, resulting in the disruption of its binding activity.  alternative splicing of cugbp2 a search of the ucsc genome browser, blat, suggested that the r3δ isoform is the product of skipping of exon in 25% of cugbp transcripts. exon is nt and the skipping transcript does not generate a new premature termination codon. to assess in which organ alternative splicing of rrm in cugbp takes place, rt-pcr was performed in adult mouse tissues. the products of exon inclusion and exon skipping, which encode a complete and partial rrm respectively, were detected. cugbp mrna was highly expressed in the brain, where the main product was the exon inclusion transcript encoding a complete rrm. the total amount of cugbp transcripts in the kidney or liver was low compared to that in brain, but the percentage of the r3δ isoform was relatively high. these results suggest that the r3δ isoform is one of major products when their gene expression of cugbp is low, and the cugbp isoform, but not r3δ, is expressed as the major product when their gene expression is high. to further investigate the alternative splicing pattern of cugbp rt-pcr was performed in p cells during neural differentiation. increased levels of the exon inclusion product were detected during neural differentiation even though the exon skipping product was not essentially changed. the r3δ percentage on day was %, which was the lowest during p neural differentiation. the relatively low expression of r3δ with respect to alternative splicing patterns was also found in tissues of the adult mouse. western blot analysis of cugbp proteins showed that the full length isoform was the main protein in p cells and that its level increased at the neural stage. by contrast, the level of r3δ decreased at the neural stage, although it was observed in undifferentiated p cells. expression analysis of cugbp target genes, actn and insulin receptor cugbp is a regulator of the alternative splicing of several transcripts, including actn and ir. the alternative splicing of actn and ir was therefore analyzed in adult mouse tissues and p cells. the actn gene has mutually exclusive exons, namely the smooth muscle exon and non-muscle exon. the sm exon., as a percentage of nm and sm exons, was higher in neural differentiated p cells than in undifferentiated p cells. in the brain, % of actn transcripts contained the sm exon, while the nm exon was predominant in the kidney and liver. cugbp has been suggested to promote the inclusion of the sm exon in prior work, suggesting that the elevated expression of cugbp and inclusion of the actn sm exon may occur in the same cells and tissues. on the other hand, the ir gene consists of exons and generates isoforms, ir-a and ir-b, which are characterized by the skipping and inclusion of exon respectively. rt-pcr analysis of the alternative splicing of the ir gene in tissues and p cells identified the two isoforms, ir-a and ir-b, in p cells during neural differentiation. although the expression of the ir gene increased during the glial cell stage, the ira/ir-b ratio did not change significantly during cell differentiation. while the exon skipping variant ir-a was expressed in the brain, the exon inclusion form ir-b was mainly expressed in the kidney and liver. because cugbp is known to repress the inclusion of ir exon, elevated expression of cugbp and the ir-a variant may occur in the same tissues, but is not observed in p cells. in addition to the expression of cugbp the ratio of r3δ to cugbp was lower in the brain than in the kidney and liver. the function of the r3δ isoform was therefore examined in detail as described in the next section. cugbp r3δ induces smooth muscle type splicing of actn1 as described previously, cugbp promotes the inclusion of the mutually exclusive sm exon instead of the nm exon in actn. the specific function of the cugbp isoform r3δ was examined by transient transfection using the mouse actn minigene. transfection of cos cells with the actn minigene alone resulted in the detection of a transcript including the nm exon as a major product and another transcript with the sm exon as a minor product. control cos cells lacking the minigene did not show these transcripts, confirming that the two transcripts containing the nm or sm exon were products of the transfected minigene. co-transfection with a myc-tagged cugbp expression vector resulted in a decrease in the nm exon product concomitant with an increase in the sm exon product. use of the etr- expression vector as a control showed that etr- promoted the use of the nm exon. these results are consistent with previous reports. to assess the function of the alternatively spliced isoform of cugbp r3δ, transient transfection experiments were performed using the actn minigene with the r3δ instead of the cugbp expression vector. r3δ induced the production of the mutually exclusive sm exon similar to cugbp, suggesting that the function of r3δ in actn splicing is similar to that of cugbp and that part of rrm which is encoded in r3δ, may be dispensable for the regulation of the splicing of actn as shown above, our results identified the brain as the location of sm exon inclusion and the kidney and liver as characteristic of nm exon inclusion. in parallel to sm exon inclusion, the expression level of cugbp was high in the brain and low in the kidney and liver, suggesting that the upregulation of cugbp in the brain contributes to the induction of sm exon inclusion. different effects of cugbp isoforms on the insulin receptor it was reported that cugbp induces the skipping of exon in ir. the induction of exon skipping by cugbp was assessed using the ir minigene in transient transfection experiments. transfection of cos or hela cells with the ir minigene alone resulted in the production of similar amounts of exon inclusion and skipping products. exon inclusion and skipping products were not detected in cos or hela cells without the minigene, confirming that they were transcribed from the transfected minigene. co-transfection of the cugbp expression vector and the ir minigene caused an increase in the exon skipping product and a decrease in the exon inclusion product. co-transfection of etr- as a control did not cause significant changes compared with the transfection of the minigene alone. these results confirmed that cugbp induces the skipping of exon as previously reported. this effect was clearer in hela cells than in cos cells. to examine potential changes in the function of the alternatively spliced isoform, r3δ, the ir minigene was co-transfected with the r3δ expression vector, which caused a decrease in exon skipping and an increase in exon inclusion in comparison to co-transfection with cugbp or the mock control. in a previous report, artificially truncated cugbp proteins with deleted n-terminal regions or deleted c-terminal regions were unable to engage in efficient exon skipping. in the present study, r3δ promoted the exon inclusion, which is in contrast to the activity to full-length cugbp which promotes exon exclusion of the ir gene. these results also indicate that part of rrm which is encoded by the alternative exon of cugbp is not only essential for the skipping of ir exon but that disruption of this part of rrm may result in a change in the splicing regulated by cugbp among celf family proteins, an isoform resembling r3δ can be predicted in celf. however, a generalized rule for rrm3s cannot be established because exon skipping can be activated by a subgroup of proteins that includes cugbp and but not by another subgroup that includes etr- or celf. our results suggest that the disruption of part of rrm in cugbp generates a new activity in the regulation of the splicing of the ir gene. because the r3δ isoform has a similar activity to cugbp in the regulation of splicing of actn it cannot be considered simply as a dominant negative isoform of cugbp the product of exon skipping was detected in the brain and the product of exon inclusion was detected in the kidney and liver, suggesting that high cugbp expression in the brain contributes to exon skipping. a high ratio of r3δ to cugbp in the kidney and liver may repress exon skipping due to the specific function of the r3δ isoform. structural analyses of the third rrm of the cugbp r3δ isoform a total of human refseq proteins with rrm domain were found in the blast. an rrm domain is usually ~ a.a. in length, and slightly smaller rrm domains, which appear to lack rnp- in the n-terminus or the dimerization module in the c-terminal region, are frequently observed in conserved domain searches of blast. rrm of cugbp consists of a.a., and a.a. of this region are disrupted in the r3δ isoform. disruption of almost half of rrm did not prevent recognition of the remaining sequence in the conserved domain search. a total of human refseq proteins with severely disrupted rrm domains are listed in table some of the domains shown in table such as eif3b, eif4b, la, u2af and u2af, have been reported to form unusual rrm structures, although the amino acid sequences were not sufficient to predict specific rrm structures. there-dimensional structures of the rrm isoforms were examined by md simulation and nmr spectroscopy. the structural analysis of the rrm of cugbp has been reported previously. several unaligned residues were found between the rrm3s of cugbp and the results of homology modeling showed that the third domain of cugbp formed an rrm structure. no significant difference could be observed between the md simulations of rrm of cugbp and rrm of cugbp. further, cugbp rrm maintained an rrm structure for ns in the md simulation. the 1h-15n hsqc spectrum of the rrm of cugbp in the absence of rna showed that, unlike rrm of cugbp the resonances of the n-terminal residues in the linker domain were concentrated in the center of the spectrum, suggesting a random coil structure. this result was probably due to the inclusion of a long n-terminal sequence in the present constructs, which forms a flexible linker region connecting rrm with rrm. protein threading and comparative modeling of the r3δ isoform were performed in the pdbj. the rrm of human cugbp was chosen as the ideal template in the search and it was predicted that the r3δ isoform with part of the linker sequence would not form an rrm structure, especially as the first α-helix was missing. we used md simulations to investigate whether the addition of linker residues to the rrm domain could restore its structure. however, even in these conditions, the first α-helix cannot be formed even after ns. moreover, resonances corresponding to the r3δ isoform were detected in the central region of the nmr spectrum, suggesting the presence of greater alterations in the structure of r3δ than those shown by md simulation and indicating that the rrm structure cannot be restored in the r3δ isoform. in addition, the interaction between cugbp or the r3δ isoform and rna molecules was examined. the addition of rna caused the disappearance of many resonances and the appearance of new resonances in cugbp. the affected residues, which are highlighted with an open box in figure 5b, were consistent with the corresponding region of cugbp and indicate that cugbp binds to in a similar manner as cugbp no spectral changes were detected in the r3δ isoform with the addition of rna, indicating that the r3δ isoform does not bind to although the rna-binding of rrm1- of cugbps is still unclear, rrm is known to be responsible for rna-binding to ug-repeats. md simulation suggested that the r3δ isoform could not form an rrm domain and the nmr spectrum showed that it did not bind to the ug-repeat. these results lead to the speculation that the disruption of the first half of rrm by exon skipping causes a defect in rna-binding. however, the r3δ isoform may interact with actn pre-mrna based on the fact that this isoform still has two rrms and two different cis-elements were reported in the alternative splicing of actn. a cugbp interacting cis-element was determined in the intronic region of ir and shown to affect the alternative splicing of ir. because this sequence is not a typical ug-repeat, perhaps the rrm may not be responsible for the primary interaction with the ir pre-mrna. therefore, other, currently unknown, proteins might interact with the rrm domain and it might be the interactions of these proteins rather than rrm rna-binding activity that is abrogated by rrm alternative splicing.  the results of the present study show that while cugbp and its r3δ isoform similarly promoted the use of the sm exon instead of the nm exon in the alternative splicing of the actn minigene, they did not similarly promote the use of exon in the alternative splicing of the ir minigene; cugbp promoted skipping of exon whereas r3δ did not. in addition, the results of md simulation and nmr suggested that the truncated rrm region in r3δ resulting from the alternative splicing of cugbp could neither form a new rrm domain nor bind to a ug-repeat. thus, it is possible that other, currently unknown, cugbp2-interacting proteins and/or other splicing factors involved in splicing regulation might be important for rrm function. our results also showed that the skipping of exon of cugbp not only disrupted cugbp rna-binding activity but also altered its splicing regulator function. a high ratio of r3δ to cugbp in the kidney and liver may affect the splicing of the ir gene and repress exon skipping. 
the present study investigated the expression of exon which is an alternatively spliced exon and encodes the first half of the third rrm of cugbp the ratio of exon skipping product to its inclusion was reduced in neuronal cells induced from p cells and in the brain. although full length cugbp and the cugbp r3δ isoforms showed a similar effect on the inclusion of the smooth muscle exon of the actn gene, these isoforms showed an opposite effect on the skipping of exon in the insulin receptor gene. our results suggest that cugbp regulates the splicing of actn and insulin receptor by different mechanisms. alternative splicing of cugbp exon contributes to the regulation of the splicing of the insulin receptor. in addition, examination of structural changes in these isoforms by molecular dynamics simulation and nmr spectrometry suggested that the third rrm of r3δ isoform was flexible and did not form an rrm structure.
 celf/bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. these rna-binding proteins contain two rna recognition motif domains at the n-terminus and another rrm at the c-terminus. cugbp is a member of this family of proteins that possesses several alternatively spliced exons.  the present study investigated the expression of exon which is an alternatively spliced exon and encodes the first half of the third rrm of cugbp the ratio of exon skipping product to its inclusion was reduced in neuronal cells induced from p cells and in the brain. although full length cugbp and the cugbp r3δ isoforms showed a similar effect on the inclusion of the smooth muscle exon of the actn gene, these isoforms showed an opposite effect on the skipping of exon in the insulin receptor gene. in addition, examination of structural changes in these isoforms by molecular dynamics simulation and nmr spectrometry suggested that the third rrm of r3δ isoform was flexible and did not form an rrm structure.  our results suggest that cugbp regulates the splicing of actn and insulin receptor by different mechanisms. alternative splicing of cugbp exon contributes to the regulation of the splicing of the insulin receptor. the present findings specifically show how alternative splicing events that result in three-dimensional structural changes in cugbp can lead to changes in its biological activity. 