BACKGROUND
rna binding proteins  are multifunctional molecules that coordinate key functions such as pre-mrna splicing, 5’ capping, 3’ end processing, nuclear-cytoplasmic transport, mrna translation and storage, and mrna processing  <cit> .
the scaffold attachment factor protein family consists of safb <dig>  safb <dig> and a rbp termed saf-like transcription modulator   <cit> .
they are large multi-domain proteins encoding a saf box , an rna recognition motif and glu/arg regions that mediate protein-protein interactions  <cit> .
scaffold attachment factor b <dig>  is an evolutionarily conserved nuclear protein that has been associated with multiple cellular processes such as transcription, chromatin structure and apoptosis  <cit> .
studies point to a role for safb <dig> in rna processing and mass-spectrometric analyses identified safb <dig> amongst approximately  <dig> proteins associated with mrna  <cit> .
safb <dig> was first identified attached to the scaffold/matrix attachment regions of chromatin  <cit> .
it was subsequently identified as het/safb <dig>  a protein that downregulates the transcription of the hsp <dig> gene  <cit>  and hnrnpa <dig> associated protein, a factor recruited to nuclear stress bodies  <cit> .
studies of safb <dig> function have shown scaffold/matrix attachment regions serve as anchors to attach chromatin to the nuclear matrix  <cit>  and the chromatin loop domains formed at these attachment regions are thought to be sites controlling transcription and replication  <cit> .
safb <dig> has also been shown to interact with proteins involved in splicing and other aspects of rna-processing, such as auf1/hnrnp d, hnrnp a <dig>  srsf <dig> , srsf <dig> , srrp <dig>  <cit> , slm- <dig>  <cit> , t-star, and sam <dig>  <cit> .
the interaction of safb <dig> with regulators of rna processing  <cit> , together with its high affinity for rna polymerase ii  <cit> , provides further evidence for its involvement in the assembly of rna regulatory complexes and alternative splicing  <cit> .
safb <dig> knockdown was shown to regulate splice site selection of the tra2β <dig> gene, further supporting a role in alternative splicing  <cit> .
safb <dig> is ubiquitously expressed in most tissues and is expressed at high levels in the developing and mature brain  <cit> .
following heat stress, safb <dig> translocates to nuclear stress bodies together with hsf <dig> and sam <dig>  it has been hypothesised that nuclear stress bodies may sequester splicing factors to downregulate normal cellular splicing and facilitate the processing of rna transcripts that are essential for the stress response  <cit> .
neurons transcribe a greater number of genes than other cell types  <cit>  and hence rbps must be expressed at high levels to coordinate the processing of rna  <cit> .
evidence also suggests that aging alters the expression and function of some rbps and this contributes to the decline in cognition and increased susceptibility to specific neurological conditions  <cit> .
safb <dig> is expressed at high levels in neurons and its expression is regulated by stress, suggesting it may play an important role in coordinating the expression of neuroprotective proteins.
surprisingly, although considerable evidence suggests safb <dig> regulates rna processing, its specific interactions with rna have not been studied.
to investigate the functional role of safb <dig> and to identify interactions with individual rnas in neuronal cells, we used individual-nucleotide resolution cross-linking and immunoprecipitation with deep sequencing   <cit> .
we found safb <dig> cross-link sites were highly enriched in exons and the purine-rich pentamers, gaaga and aagaa, are the most significantly enriched motifs within the binding sites.
further analyses using high-resolution exon junction arrays showed there were significant changes in the splicing patterns of a number of genes following safb <dig> knockdown.
in addition, we analysed the frequency of predicted safb <dig> target motifs  <cit>  and found that there was a significant increase in the occurrence of paired trimer motifs within the last  <dig> nt of exons downregulated following safb <dig> knockdown.
the results also showed that safb <dig> regulated splicing within an ncam <dig> minigene but this regulation was lost when the aga, gaa, aag recognition motifs were mutated.
ncam <dig> and many rnas bound by safb <dig> have roles in neuronal plasticity and function.
furthermore, empirical analyses showed that safb <dig> regulated dendritic spine size in hippocampal neurons, confirming that safb <dig> plays an important role in coordinating gene processing in neurons.
RESULTS
safb <dig> iclip analyses
the sequences of safb <dig> and safb <dig> share considerable identity and we therefore assessed the specificity of the anti-safb <dig> antibody.
the results  showed that the safb <dig> antibody detected a reduction in safb <dig> expression following the sirna-mediated knockdown of safb <dig> but not following the knockdown of safb <dig>  in addition, the safb <dig> antibody did not detect enhanced green fluorescence protein -tagged safb <dig> when expressed exogenously by an adenovirus .
following confirmation of antibody-specificity, iclip experiments were carried out in triplicate on sh-sy5y neuroblastoma cells.
cells were uv-crosslinked to mediate the formation of protein-rna complexes and the protocol validated using cell extracts from non- crosslinked cultures  and those transduced with an adenoviral vector expressing safb <dig>  in the absence of uv crosslinking, 32p-end-labelled rna was not immunoprecipitated with safb <dig> .
iclip was performed with partial rnase i digestion and, following sds-page analysis of immunoprecipitated safb <dig>  the cross-linked rna was extracted, reverse-transcribed and pcr-amplified before being subjected to high-throughput sequencing using illumina ga <dig>  removing sequences that truncated at the same nucleotide in the genome and shared the same random barcode eliminated pcr amplification artefacts.
this left a total of  <dig>  million unique sequence reads that aligned to the human genome hg <dig>  allowing only a single nucleotide mismatch, where each read represents a single cdna molecule.
in the no antibody control, ~ <dig>   reads were found.
the distribution of the iclip-tags to rna was analysed  and mapped to open reading frames, intergenic and intronic regions, non-coding rna , and untranslated regions  .
we also mapped the fold enrichment of clip-tag density  in different rnas relative to the average clip-tag density in the genome .
the tags were highly enriched in open reading frames , 3’ and 5’ utrs , and non-coding rnas.
within genes, the highest densities of safb <dig> cross-link sites  were located in exons in proximity to intron-exon and exon-intron boundaries .
safb <dig> cross-link sites fall predominantly within exons, suggesting a role in pre-mrna splicing or in the fate of mrnps after splicing.
to investigate the rna sequence specificity of safb <dig>  the z-scores of possible pentamers within the 61-nucleotide sequence surrounding all cross-link sites  were calculated by plotting the experimental repeats against each other .
in each case, the gaaga motif was found to be the most significantly enriched .
we also included the z-scores  obtained from iclip experiments investigating the interaction of safb <dig> under stress conditions.
when these data were analysed together, the most significantly enriched pentamers were again gaaga and aagaa  .
these data also identify the gaa, aag and aga trimers as the core motifs likely to be recognised by safb <dig>  <cit> .
to assess the potential functions of safb <dig>  a gene ontology  analysis of the rnas with greater than  <dig> tags  was carried out and the most significant biological processes are shown .
safb <dig> was predicted to be involved in the expression of proteins involved in chromosome organisation, rna processing , the cellular response to stress, and neuron projection and morphogenesis.fig.
<dig> analyses of safb <dig> rna-interactions by iclip.
a western blots showing that following sirna-mediated knockdown of safb <dig> and safb <dig>  the safb <dig> ab detects a reduction in endogenous safb <dig> but not endogenous safb <dig> and neither does the ab recognize safb <dig> when mediated by an adenovirus expressing egfp-tagged safb <dig>  conversely, the safb <dig> antibody  shows a reduction in endogenous safb <dig> expression following treatment with the safb <dig> sirna and also detects the egfp tagged safb <dig> protein.
b the bar graphs show the proportion of cdnas from the iclip experiments and their mapping to different genomic regions   and normalized iclip read densities  mapped to different genomic regions.
d safb <dig> crosslink sites mapped around the 3’ and 5’ splice sites from − <dig> to + <dig> nucleotides with the splice junction at position  <dig>  data are normalized by all junctions spanning position, smoothed and represented as 109 × /.
e z-scores of pentamer occurrence within the 61-nt sequence surrounding crosslink sites calculated by plotting data from individual iclip experiments .
the most enriched pentamers are shown and safb <dig> recognized the gaaga pentamer with the highest frequency
real-time pcr analysis of functionally-related genes of interest , and astn <dig> and pde4b ) with iclip crosslinks was performed to investigate a functional interaction.
three of these genes  contained greater than  <dig> tags , while ncam <dig> contained  <dig>  total rna was isolated from sh-sy5y cells following sirna-mediated safb <dig> knockdown , reverse-transcribed and pcr-amplified .
several specific regulatory changes were identified in this way, for instance, safb <dig> knockdown significantly altered the level of specific ncam <dig> and synpo <dig> isoforms.
these results are suggestive of an effect of safb <dig> on alternative splicing since different splicing patterns are used to generate these isoforms.
iclip tags found in tra2β <dig> further support safb1’s role in alternative splicing, as splice site selection for this gene is regulated by safb <dig>  <cit> .
the expression of astn <dig> and pde4b was also altered by safb <dig> knockdown with increased and decreased isoform expression being noted likely due to effects of safb <dig> on alternative promoters.
furthermore, the expression of genes found to have a high number of safb <dig> tags  were also found to be significantly altered following safb <dig> knockdown .
safb <dig> tags were also found in the genes of non-coding rnas so the effect of safb <dig>  knockdown on the processing of mature mirnas from the mir17hg  mirna cluster was analysed further.
we found that both safb <dig> and safb <dig> knockdown mediated a decrease in the expression/processing of mir19a, but no effect on mir- <dig> or mir- <dig> was noted .
the simultaneous knockdown of safb <dig> and safb <dig> also mediated a significant decrease in the expression of mir-19a.
in addition, rnaseq experiments showed safb <dig> knockdown significantly increased expression of the primary mir-17- <dig> transcript .
taken together, these results validate the iclip results and show that safb <dig> is interacting with specific coding rna transcripts to modulate transcript expression and possibly regulate alternative splicing.fig.
<dig> safb <dig> knockdown mediates changes in the expression of specific rna isoforms.
rt-pcr analysis of genes with crosslinks identified by iclip was performed in such a way as to examine the possibility of transcript-specific alterations.
a rna was isolated from shsy5y cells following sirna mediated safb <dig> knockdown and safb <dig> and safb <dig> rna levels measured.
b specific primer sets were designed and used to measure the expression of ncam <dig>  synpo <dig>  astn <dig> and pde4b isoforms.
the results were obtained following three independent experiments.
statistical analysis was by anova followed by post-hoc t-tests.
*p < <dig> , **p < <dig>
fig.
<dig> safb <dig> regulates mir17hg.
a schematic representation of the mirna encoded in the mir17hg .
b knockdown of safb <dig> mediates a decrease in the level of mir-19a.
means ± sd are shown.
anova followed by post-hoc t-tests.
*p < <dig>
investigating the regulation of alternative splicing by safb <dig> using ncam <dig> minigenes
the iclip analysis suggested that safb <dig> may play a role in alternative splicing; to investigate this further, we used a ncam <dig> minigene.
we chose to use ncam <dig> as  both safb <dig> and ncam <dig> are highly-expressed in the hippocampus and a significant body of work has shown that ncam <dig> is an important regulator of synaptic function  <cit> , and its altered expression  <cit>  and splicing  <cit>  is implicated in the aetiology of human neuropsychiatric illnesses; and  since safb <dig> iclip tags were enriched over exon  <dig> , it was appropriate to construct a wild type ncam <dig> minigene with exons  <dig>   <dig>   <dig> and  <dig>  and a mutant minigene in which mutations were introduced to alter all aag/aga/gaa trimers in exon  <dig> .
using rt-pcr and qpcr analyses  we found that expression of the ncam <dig> 9– <dig> isoform was significantly reduced following safb <dig> knockdown .
this effect was abolished when trimers were mutated in exon  <dig>  supporting the notion that direct binding of safb <dig> to exon  <dig> enhances expression of the 9– <dig> isoform from the minigene .
regulation of expression of the ncam 7– <dig> and 8– <dig> isoforms appears to be somewhat more complicated, with mutation of trimers in exon  <dig> making expression of these isomers more dependent on safb <dig>  the latter results suggest that safb <dig> might be enhancing expression of the 7– <dig> and 8– <dig> isoforms by binding elsewhere in the minigene, or perhaps via an indirect effect.fig.
<dig> safb <dig> regulates splicing from wild type and mutant ncam <dig> minigenes.
a the distribution of safb <dig> iclip tags within the ncam <dig> minigene is shown and an arrow indicates the position of exon  <dig> in transcript variant  <dig>  b the schematic indicates the composition of the minigenes and splice forms generated.
the sequence of wild type exon  <dig>  the location of the gaa/aag and aga motifs, and the mutations introduced to alter all aag/aga/gaa are shown.
c pcr analyses using exon-specific spanning primers was used to analyse the effect of safb <dig> knockdown on the expression of the ncam <dig> isoforms  in the wild type and mutant minigenes.
the open bars represent expression of the transcript indicated with non-targeting control knockdown and the closed bars following safb <dig> knockdown.
values are means ± sem, statistical analysis was performed by anova and post-hoc t-tests *p < <dig> , **p < <dig> , ***p < <dig>
exon microarray analysis
to further investigate the association of safb <dig> with splicing, microarray analysis with a human exon  <dig>  st affymetrix array was used .
triplicate samples were prepared from sysy5y cells following sirna-mediated safb <dig> knockdown and transfection with a sirna non-targeting control.
we used altanalyse   <cit>  to interrogate the microarray data for alternative exon expression.
following safb <dig> knockdown, we found  <dig> exons were significantly downregulated and  <dig> exons were upregulated .
summaries of the significant positive and negative changes found by altanalyse are shown in fig.
5a, which show that safb <dig> can promote and silence expression of rna elements.
next, we confirmed these results using rt-pcr to assess  <dig> splicing events identified by arrays.
primer pairs were generated to distinguish isoforms, and  <dig> of these reproduced the direction of the observed splicing change, resulting in a  <dig> %  validation rate of the microarray data .
we also investigated the effects of safb <dig> on non-coding transcripts.
safb <dig> was found to have a very high number of cross-link sites across the long non-coding rna  malat <dig> but not in the area that encodes the cleaved masc <dig> transcript.
further experiments using malat1-specific pcr showed that safb <dig> knockdown significantly increased the expression of malat <dig> .
we also conducted a go analysis of genes that the exon array identified as being differentially expressed  following safb knockdown .
this analysis linked safb <dig> interactions with the expression of genes associated with meiosis, the cytoskeleton, and/or involved with atp/nucleoside binding, cytoskeletal organisation and microtubule-based processes.fig.
<dig> exon array analyses following safb <dig> knockdown.
samples were prepared from sysy5y cells following sirna-mediated safb <dig> knockdown and  <dig>  st affymetrix high-resolution exon arrays used to analyse effects on splicing.
altanalyse was used to interrogate the microarray data for alternative splicing events and the significant changes are shown .
rt-pcr was used to confirm the splicing events identified by arrays .
primer pairs were generated to distinguish isoforms and three independent experiments were carried out in triplicate.
safb <dig> knockdown mediated statistically significant changes in expression of the gene transcripts shown in .
statistical analysis was by anova followed by post-hoc t-tests.
**p < <dig> , *p < <dig> .
b lane  <dig> control; lane  <dig> safb <dig> knockdown.
open bars in  represent controls, filled bars represent safb <dig> knockdown
distribution of motifs recognised by safb <dig> using rnamotifs
recently, the clustering property of specific elements/motifs  motivated the development of the rnamotifs software that identifies multivalent motifs predicted to regulate splicing  <cit> .
this method assumes most rna-binding domains recognise a core of up to four nucleotides and our iclip analyses suggest the gaa/aag/aga nucleotide sequences form the core motifs recognised by safb <dig>  as the core motifs were already predicted by iclip analyses, we modified the rnamotifs approach to analyse the distribution of these  trimers within exons differentially expressed following safb <dig> knockdown .
we initially looked at the proportion of differentially expressed exons  that contained the trimers of interest at particular locations in upregulated and downregulated exons compared to our control exons.
in the first  <dig> nt of differentially expressed upregulated and downregulated exons we did note differences compared to controls.
however, there was an enrichment of the safb <dig> trimer motifs in the last  <dig> nt of downregulated cassette exons, indicating that there may be increased inclusion of the cassette exon in the presence of safb <dig>  to obtain an approximate false discovery rate  for these hits, we used a bootstrapping approach that selected exons randomly from the genome, counted pair motifs in each exon, and then compared the counts with our control set.
in each trial we sampled  <dig> exons without replacement from a population of  <dig>  known exons and used fisher’s exact test to compare the frequencies of each motif pair with the frequencies in the control set.
the fdr was then given by bootstrapping  <dig>  samples and p values greater than  <dig>  were considered not significant .
by far the most significant pair motif was aga paired with any of the three trimers  found downstream of it .
this result is consistent with those made by cereda et al.
<cit> , in that it suggests rbps bind multivalent patterns to modulate splicing.fig.
<dig> distribution of differentially expressed purine-rich trimer exons following safb <dig> knockdown.
bar graphs showing the proportion of exons that contain the trimers of interest at particular locations in upregulated  and downregulated  exons compared to controls.
the table shows the most significant motif pairs found in the last  <dig> nt of downregulated alternative exons for the three trimers  along with degenerate pairs defined as a specific target trimer paired with aag, aag or aga .
shown are the most highly significant pairings over-represented in downregulated exons, .
bootstrapping with  <dig>  samples yielded the estimated false discovery rates
safb <dig> expression and function in neurons
the ncam <dig> minigene experiments together with the iclip and exon array analyses suggest safb <dig> may play an important role in regulating the organisation of the cytoskeleton, neuronal development and synaptic function.
immunocytochemical analyses showed that safb <dig> expression was evident throughout the brain but was most pronounced in hippocampal neurons .
therefore, to investigate effects of safb <dig> on indices of synaptic function we transduced primary hippocampal neurons that had been  <dig> days in culture  with an adenovirus expressing safb <dig>  four days later, we fixed the cells and used confocal microscopy to analyse spine number and size .
the results showed that safb <dig> expression significantly increased spine size  but had no effect on spine number .fig.
<dig> safb <dig> regulated dendritic spine size in primary hippocampal neurons.
a immunocytochemical analyses show safb <dig> is highly expressed in the hippocampus .
safb <dig> is clearly evident in pyramidal neuron cell bodies in the ca <dig> region of the hippocampus  red safb <dig> immunofluorescence) and a neurofilament stain of dendrites  second panel) shows safb <dig> is located exclusively in neuronal cell bodies.
primary hippocampal neurons were transduced with safb-egfp or egfp alone  and confocal microscopy used to measure spine  area,  size, and  density.
spine size measures were analysed by cumulative frequency using the kolmogorov-smirnov test  and by t-test  ***p < <dig> .
values shown in bar graphs are means ± sd
discussion
following transcription, rna transcripts immediately associate with proteins that facilitate their processing and export.
safb <dig>  can bind rna via its n-terminal rna recognition motifs; however, these interactions have not yet been investigated using genomic profiling techniques.
we used the powerful iclip-seq method to identify rna targets of safb <dig> and our results showed it interacted with hundreds of rna species with the greatest enrichment being in exons, non-coding rnas, and 5’ and 3’ utrs.
safb <dig> binding to exons was associated with the purine-rich pentamers, gaaga and aagaa, that occurred with the highest frequency in proximity to intron-exon and exon-intron boundaries.
go analyses based on these interactions suggested safb <dig> was likely to have roles in regulating expression of proteins involved in rna processing , the cellular response to stress, and neuron projection and morphogenesis.
ncam <dig>  synpo <dig>  astn and pde4b are examples of genes with multiple safb <dig> tags and further investigation, including ncam <dig> minigene experiments , confirmed safb <dig> knockdown did alter their expression.
interestingly, ncam <dig>  <cit> , astn <dig>  <cit> , and pde4b  <cit>  are known to play important roles in regulating synaptic function and are implicated in human psychiatric disease.
importantly, safb <dig> was also found to regulate the processing of non-coding rnas.
safb <dig> has multiple cross-link sites along the lncrna malat <dig> and safb <dig> knockdown increased malat <dig> expression.
malat <dig> has been shown to interact with multiple sr proteins  to regulate alternative splicing.
previous studies using iclip have shown that the splice regulators tdp- <dig>  srsf, srsf <dig> and srsf <dig> also cross-link extensively with malat <dig>  and this interaction may therefore be common with rbps that regulate splicing.
iclip-seq analyses also showed that safb <dig> interacted with the physiologically important mir-17- <dig> mirna cluster  <cit> .
we further showed that this interaction had functional consequences, with safb <dig> knockdown being found to significantly increase the expression of the mir-17- <dig> primary transcript and reduce the expression of mir-19a.
interestingly, and related to the functions predicted by the go analyses, the mir-17- <dig> cluster  has been shown to be an important regulator of axonal function  <cit> .
furthermore, mir-17- <dig> cluster dysregulation has been implicated in a number of cancers  <cit> , human aging and in neurodegenerative disease  <cit> .
in this study, we validated interactions with genes that had a high and comparatively low number of safb <dig> tags suggesting the presence of iclip tags was a good predictor of functionally important interactions.
to further investigate the role safb <dig> plays in alternative splicing we used rnamotifs to analyse the enrichment and distribution of the gaa, aag and aga binding motifs in differentially expressed exons.
this analysis allowed position-dependent effects on exon inclusion/exclusion following safb <dig> binding to be predicted.
the frequency of one or more motif pairs  was significantly increased within downregulated exons suggesting safb <dig> in these cases promoted exon inclusion.
safb <dig> iclip tags were found in the ncam <dig> gene and ncam <dig> minigene experiments showed that safb <dig> knockdown altered alternative splicing and that mutation of the gaa, aag and aga motifs significantly decreased this regulation.
these data therefore support the iclip and rnamotifs analyses, which together suggested safb <dig> regulates the alternative splicing of specific genes via recognition of ag rich motifs.
safb <dig> has been implicated in the regulation of a number of cellular processes; however, as it is a multi-domain protein defining specific roles has proved difficult.
the regulation of ncam <dig> splicing and its interaction with known splicing regulators and with polymerase ii suggests it may be involved in spliceosome assembly.
we provide evidence supporting this hypothesis and suggest that safb <dig> may act to recruit splicing factors to a small number of pre-mrnas.
the dead-box helicase eif4aiii, a core component of the exon junction complex  was found to bind within exon regions rich in gaaga motifs at sites distal and close to ejc regions  <cit> .
as well as safb <dig> and eif4aiii, a number of sr proteins have been found to bind the n motif, including srsf <dig> , htra <dig> and srm <dig>  <cit> .
it was suggested that gaaga might be a high-affinity binding site for factors that influence ejc binding; as such, it is possible that these proteins bind this motif to attract ejc factors during splicing and/or to stabilize the ejc in specific genes.
safb <dig> expression levels differ between tissues and, as its expression levels in  neurons  are high, it is therefore possible that safb <dig> plays a more significant role in regulating rna processing in these tissues.
the level and sub-cellular location of safb <dig> also differs following stress and this may also influence the degree to which safb <dig> competes with other  proteins for the recognition site.
similarly, as safb <dig> is known to bind other splice regulators, its expression levels may also influence the sequence preference/specificity of spliceosome complexes and thereby regulate cellular function.
go functional analyses  suggested safb <dig> regulates the expression of proteins involved in atp/nucleoside binding, cytoskeletal organisation and microtubule-based processes.
importantly, minigene experiments showed the alternative splicing of ncam <dig> was altered by safb <dig>  ncam <dig> is an important regulator of synaptic function  <cit>  and its altered expression  <cit>  and splicing  <cit>  are implicated in the aetiology of human neuropsychiatric illnesses.
safb <dig> mediated isoform-specific changes in ank <dig>  <cit> , anks1b  <cit> , sap <dig>   <cit> , add <dig>  <cit> , kif16b  <cit> , and elk <dig>  <cit> , as well as in genes linked to the cytoskeleton and/or synaptic function and the aetiology of neurologic disease.
for example, the ankyrin-g protein encoded by ank <dig> organizes synaptic microtubules and is involved in protein trafficking, neurogenesis and neuroprotection, and in the aetiology of bipolar disorder and schizophrenia  <cit> .
safb <dig> is located at 19p <dig> -p <dig>  and separate studies have found snps or duplications at this locus are associated with increased susceptibility to schizophrenia .
these data  suggest that altered function of safb <dig> could result in changes in the expression of genes that regulate synaptic function, some of which are also susceptibility genes for schizophrenia.
CONCLUSIONS
our global analysis shows safb <dig> interacts with distinct classes of rna indicating it has multiple functions with regard to rna processing and the control of gene expression and function.
importantly, we confirmed it binds primarily within exons and that safb1’s altered expression regulates the alternative splicing of genes with important roles in neuronal function .
iclip data and further analysis using rnamotifs predicted that safb <dig> is likely to interact with specific purine-rich trimer sequences to promote exon inclusion.
the safb family consists of safb <dig>  safb <dig> and sltm, proteins that share considerable identity  and are all highly but differentially expressed in the central nervous system.
the safb proteins will therefore have novel and unexplored roles in regulating the processing of coding and non-coding pre-mrnas in the brain and, as such, represent a new and important family of rna regulators.

 rna binding proteins are multifunctional molecules that coordinate key functions such as pre-mrna splicing, 5’ capping, 3’ end processing, nuclear-cytoplasmic transport, mrna translation and storage, and mrna processing. the scaffold attachment factor protein family consists of safb safb and a rbp termed saf-like transcription modulator. they are large multi-domain proteins encoding a saf box, an rna recognition motif and glu/arg regions that mediate protein-protein interactions. scaffold attachment factor b is an evolutionarily conserved nuclear protein that has been associated with multiple cellular processes such as transcription, chromatin structure and apoptosis. studies point to a role for safb in rna processing and mass-spectrometric analyses identified safb amongst approximately proteins associated with mrna. safb was first identified attached to the scaffold/matrix attachment regions of chromatin. it was subsequently identified as het/safb a protein that downregulates the transcription of the hsp gene and hnrnpa associated protein, a factor recruited to nuclear stress bodies. studies of safb function have shown scaffold/matrix attachment regions serve as anchors to attach chromatin to the nuclear matrix and the chromatin loop domains formed at these attachment regions are thought to be sites controlling transcription and replication. safb has also been shown to interact with proteins involved in splicing and other aspects of rna-processing, such as auf1/hnrnp d, hnrnp a srsf, srsf, srrp, slm-, t-star, and sam. the interaction of safb with regulators of rna processing, together with its high affinity for rna polymerase ii, provides further evidence for its involvement in the assembly of rna regulatory complexes and alternative splicing. safb knockdown was shown to regulate splice site selection of the tra2β gene, further supporting a role in alternative splicing. safb is ubiquitously expressed in most tissues and is expressed at high levels in the developing and mature brain. following heat stress, safb translocates to nuclear stress bodies together with hsf and sam it has been hypothesised that nuclear stress bodies may sequester splicing factors to downregulate normal cellular splicing and facilitate the processing of rna transcripts that are essential for the stress response. neurons transcribe a greater number of genes than other cell types and hence rbps must be expressed at high levels to coordinate the processing of rna. evidence also suggests that aging alters the expression and function of some rbps and this contributes to the decline in cognition and increased susceptibility to specific neurological conditions. safb is expressed at high levels in neurons and its expression is regulated by stress, suggesting it may play an important role in coordinating the expression of neuroprotective proteins. surprisingly, although considerable evidence suggests safb regulates rna processing, its specific interactions with rna have not been studied. to investigate the functional role of safb and to identify interactions with individual rnas in neuronal cells, we used individual-nucleotide resolution cross-linking and immunoprecipitation with deep sequencing. we found safb cross-link sites were highly enriched in exons and the purine-rich pentamers, gaaga and aagaa, are the most significantly enriched motifs within the binding sites. further analyses using high-resolution exon junction arrays showed there were significant changes in the splicing patterns of a number of genes following safb knockdown. in addition, we analysed the frequency of predicted safb target motifs and found that there was a significant increase in the occurrence of paired trimer motifs within the last nt of exons downregulated following safb knockdown. the results also showed that safb regulated splicing within an ncam minigene but this regulation was lost when the aga, gaa, aag recognition motifs were mutated. ncam and many rnas bound by safb have roles in neuronal plasticity and function. furthermore, empirical analyses showed that safb regulated dendritic spine size in hippocampal neurons, confirming that safb plays an important role in coordinating gene processing in neurons.  safb iclip analyses the sequences of safb and safb share considerable identity and we therefore assessed the specificity of the anti-safb antibody. the results showed that the safb antibody detected a reduction in safb expression following the sirna-mediated knockdown of safb but not following the knockdown of safb in addition, the safb antibody did not detect enhanced green fluorescence protein -tagged safb when expressed exogenously by an adenovirus. following confirmation of antibody-specificity, iclip experiments were carried out in triplicate on sh-sy5y neuroblastoma cells. cells were uv-crosslinked to mediate the formation of protein-rna complexes and the protocol validated using cell extracts from non- crosslinked cultures and those transduced with an adenoviral vector expressing safb in the absence of uv crosslinking, 32p-end-labelled rna was not immunoprecipitated with safb. iclip was performed with partial rnase i digestion and, following sds-page analysis of immunoprecipitated safb the cross-linked rna was extracted, reverse-transcribed and pcr-amplified before being subjected to high-throughput sequencing using illumina ga removing sequences that truncated at the same nucleotide in the genome and shared the same random barcode eliminated pcr amplification artefacts. this left a total of million unique sequence reads that aligned to the human genome hg allowing only a single nucleotide mismatch, where each read represents a single cdna molecule. in the no antibody control, ~ reads were found. the distribution of the iclip-tags to rna was analysed and mapped to open reading frames, intergenic and intronic regions, non-coding rna, and untranslated regions. we also mapped the fold enrichment of clip-tag density in different rnas relative to the average clip-tag density in the genome. the tags were highly enriched in open reading frames, 3’ and 5’ utrs, and non-coding rnas. within genes, the highest densities of safb cross-link sites were located in exons in proximity to intron-exon and exon-intron boundaries. safb cross-link sites fall predominantly within exons, suggesting a role in pre-mrna splicing or in the fate of mrnps after splicing. to investigate the rna sequence specificity of safb the z-scores of possible pentamers within the 61-nucleotide sequence surrounding all cross-link sites were calculated by plotting the experimental repeats against each other. in each case, the gaaga motif was found to be the most significantly enriched. we also included the z-scores obtained from iclip experiments investigating the interaction of safb under stress conditions. when these data were analysed together, the most significantly enriched pentamers were again gaaga and aagaa. these data also identify the gaa, aag and aga trimers as the core motifs likely to be recognised by safb. to assess the potential functions of safb a gene ontology analysis of the rnas with greater than tags was carried out and the most significant biological processes are shown. safb was predicted to be involved in the expression of proteins involved in chromosome organisation, rna processing, the cellular response to stress, and neuron projection and morphogenesis.fig. analyses of safb rna-interactions by iclip. a western blots showing that following sirna-mediated knockdown of safb and safb the safb ab detects a reduction in endogenous safb but not endogenous safb and neither does the ab recognize safb when mediated by an adenovirus expressing egfp-tagged safb conversely, the safb antibody shows a reduction in endogenous safb expression following treatment with the safb sirna and also detects the egfp tagged safb protein. b the bar graphs show the proportion of cdnas from the iclip experiments and their mapping to different genomic regions and normalized iclip read densities mapped to different genomic regions. d safb crosslink sites mapped around the 3’ and 5’ splice sites from − to + nucleotides with the splice junction at position data are normalized by all junctions spanning position, smoothed and represented as 109 × /. e z-scores of pentamer occurrence within the 61-nt sequence surrounding crosslink sites calculated by plotting data from individual iclip experiments. the most enriched pentamers are shown and safb recognized the gaaga pentamer with the highest frequency real-time pcr analysis of functionally-related genes of interest, and astn and pde4b ) with iclip crosslinks was performed to investigate a functional interaction. three of these genes contained greater than tags, while ncam contained total rna was isolated from sh-sy5y cells following sirna-mediated safb knockdown, reverse-transcribed and pcr-amplified. several specific regulatory changes were identified in this way, for instance, safb knockdown significantly altered the level of specific ncam and synpo isoforms. these results are suggestive of an effect of safb on alternative splicing since different splicing patterns are used to generate these isoforms. iclip tags found in tra2β further support safb1’s role in alternative splicing, as splice site selection for this gene is regulated by safb. the expression of astn and pde4b was also altered by safb knockdown with increased and decreased isoform expression being noted likely due to effects of safb on alternative promoters. furthermore, the expression of genes found to have a high number of safb tags were also found to be significantly altered following safb knockdown. safb tags were also found in the genes of non-coding rnas so the effect of safb knockdown on the processing of mature mirnas from the mir17hg mirna cluster was analysed further. we found that both safb and safb knockdown mediated a decrease in the expression/processing of mir19a, but no effect on mir- or mir- was noted. the simultaneous knockdown of safb and safb also mediated a significant decrease in the expression of mir-19a. in addition, rnaseq experiments showed safb knockdown significantly increased expression of the primary mir-17- transcript. taken together, these results validate the iclip results and show that safb is interacting with specific coding rna transcripts to modulate transcript expression and possibly regulate alternative splicing.fig. safb knockdown mediates changes in the expression of specific rna isoforms. rt-pcr analysis of genes with crosslinks identified by iclip was performed in such a way as to examine the possibility of transcript-specific alterations. a rna was isolated from shsy5y cells following sirna mediated safb knockdown and safb and safb rna levels measured. b specific primer sets were designed and used to measure the expression of ncam synpo astn and pde4b isoforms. the results were obtained following three independent experiments. statistical analysis was by anova followed by post-hoc t-tests. *p <, **p < fig. safb regulates mir17hg. a schematic representation of the mirna encoded in the mir17hg. b knockdown of safb mediates a decrease in the level of mir-19a. means ± sd are shown. anova followed by post-hoc t-tests. *p < investigating the regulation of alternative splicing by safb using ncam minigenes the iclip analysis suggested that safb may play a role in alternative splicing; to investigate this further, we used a ncam minigene. we chose to use ncam as both safb and ncam are highly-expressed in the hippocampus and a significant body of work has shown that ncam is an important regulator of synaptic function, and its altered expression and splicing is implicated in the aetiology of human neuropsychiatric illnesses; and since safb iclip tags were enriched over exon, it was appropriate to construct a wild type ncam minigene with exons and and a mutant minigene in which mutations were introduced to alter all aag/aga/gaa trimers in exon. using rt-pcr and qpcr analyses we found that expression of the ncam 9– isoform was significantly reduced following safb knockdown. this effect was abolished when trimers were mutated in exon supporting the notion that direct binding of safb to exon enhances expression of the 9– isoform from the minigene. regulation of expression of the ncam 7– and 8– isoforms appears to be somewhat more complicated, with mutation of trimers in exon making expression of these isomers more dependent on safb the latter results suggest that safb might be enhancing expression of the 7– and 8– isoforms by binding elsewhere in the minigene, or perhaps via an indirect effect.fig. safb regulates splicing from wild type and mutant ncam minigenes. a the distribution of safb iclip tags within the ncam minigene is shown and an arrow indicates the position of exon in transcript variant b the schematic indicates the composition of the minigenes and splice forms generated. the sequence of wild type exon the location of the gaa/aag and aga motifs, and the mutations introduced to alter all aag/aga/gaa are shown. c pcr analyses using exon-specific spanning primers was used to analyse the effect of safb knockdown on the expression of the ncam isoforms in the wild type and mutant minigenes. the open bars represent expression of the transcript indicated with non-targeting control knockdown and the closed bars following safb knockdown. values are means ± sem, statistical analysis was performed by anova and post-hoc t-tests *p <, **p <, ***p < exon microarray analysis to further investigate the association of safb with splicing, microarray analysis with a human exon st affymetrix array was used. triplicate samples were prepared from sysy5y cells following sirna-mediated safb knockdown and transfection with a sirna non-targeting control. we used altanalyse to interrogate the microarray data for alternative exon expression. following safb knockdown, we found exons were significantly downregulated and exons were upregulated. summaries of the significant positive and negative changes found by altanalyse are shown in fig. 5a, which show that safb can promote and silence expression of rna elements. next, we confirmed these results using rt-pcr to assess splicing events identified by arrays. primer pairs were generated to distinguish isoforms, and of these reproduced the direction of the observed splicing change, resulting in a % validation rate of the microarray data. we also investigated the effects of safb on non-coding transcripts. safb was found to have a very high number of cross-link sites across the long non-coding rna malat but not in the area that encodes the cleaved masc transcript. further experiments using malat1-specific pcr showed that safb knockdown significantly increased the expression of malat. we also conducted a go analysis of genes that the exon array identified as being differentially expressed following safb knockdown. this analysis linked safb interactions with the expression of genes associated with meiosis, the cytoskeleton, and/or involved with atp/nucleoside binding, cytoskeletal organisation and microtubule-based processes.fig. exon array analyses following safb knockdown. samples were prepared from sysy5y cells following sirna-mediated safb knockdown and st affymetrix high-resolution exon arrays used to analyse effects on splicing. altanalyse was used to interrogate the microarray data for alternative splicing events and the significant changes are shown. rt-pcr was used to confirm the splicing events identified by arrays. primer pairs were generated to distinguish isoforms and three independent experiments were carried out in triplicate. safb knockdown mediated statistically significant changes in expression of the gene transcripts shown in. statistical analysis was by anova followed by post-hoc t-tests. **p <, *p <. b lane control; lane safb knockdown. open bars in represent controls, filled bars represent safb knockdown distribution of motifs recognised by safb using rnamotifs recently, the clustering property of specific elements/motifs motivated the development of the rnamotifs software that identifies multivalent motifs predicted to regulate splicing. this method assumes most rna-binding domains recognise a core of up to four nucleotides and our iclip analyses suggest the gaa/aag/aga nucleotide sequences form the core motifs recognised by safb as the core motifs were already predicted by iclip analyses, we modified the rnamotifs approach to analyse the distribution of these trimers within exons differentially expressed following safb knockdown. we initially looked at the proportion of differentially expressed exons that contained the trimers of interest at particular locations in upregulated and downregulated exons compared to our control exons. in the first nt of differentially expressed upregulated and downregulated exons we did note differences compared to controls. however, there was an enrichment of the safb trimer motifs in the last nt of downregulated cassette exons, indicating that there may be increased inclusion of the cassette exon in the presence of safb to obtain an approximate false discovery rate for these hits, we used a bootstrapping approach that selected exons randomly from the genome, counted pair motifs in each exon, and then compared the counts with our control set. in each trial we sampled exons without replacement from a population of known exons and used fisher’s exact test to compare the frequencies of each motif pair with the frequencies in the control set. the fdr was then given by bootstrapping samples and p values greater than were considered not significant. by far the most significant pair motif was aga paired with any of the three trimers found downstream of it. this result is consistent with those made by cereda et al., in that it suggests rbps bind multivalent patterns to modulate splicing.fig. distribution of differentially expressed purine-rich trimer exons following safb knockdown. bar graphs showing the proportion of exons that contain the trimers of interest at particular locations in upregulated and downregulated exons compared to controls. the table shows the most significant motif pairs found in the last nt of downregulated alternative exons for the three trimers along with degenerate pairs defined as a specific target trimer paired with aag, aag or aga. shown are the most highly significant pairings over-represented in downregulated exons,. bootstrapping with samples yielded the estimated false discovery rates safb expression and function in neurons the ncam minigene experiments together with the iclip and exon array analyses suggest safb may play an important role in regulating the organisation of the cytoskeleton, neuronal development and synaptic function. immunocytochemical analyses showed that safb expression was evident throughout the brain but was most pronounced in hippocampal neurons. therefore, to investigate effects of safb on indices of synaptic function we transduced primary hippocampal neurons that had been days in culture with an adenovirus expressing safb four days later, we fixed the cells and used confocal microscopy to analyse spine number and size. the results showed that safb expression significantly increased spine size but had no effect on spine number.fig. safb regulated dendritic spine size in primary hippocampal neurons. a immunocytochemical analyses show safb is highly expressed in the hippocampus. safb is clearly evident in pyramidal neuron cell bodies in the ca region of the hippocampus red safb immunofluorescence) and a neurofilament stain of dendrites second panel) shows safb is located exclusively in neuronal cell bodies. primary hippocampal neurons were transduced with safb-egfp or egfp alone and confocal microscopy used to measure spine area, size, and density. spine size measures were analysed by cumulative frequency using the kolmogorov-smirnov test and by t-test ***p <. values shown in bar graphs are means ± sd discussion following transcription, rna transcripts immediately associate with proteins that facilitate their processing and export. safb can bind rna via its n-terminal rna recognition motifs; however, these interactions have not yet been investigated using genomic profiling techniques. we used the powerful iclip-seq method to identify rna targets of safb and our results showed it interacted with hundreds of rna species with the greatest enrichment being in exons, non-coding rnas, and 5’ and 3’ utrs. safb binding to exons was associated with the purine-rich pentamers, gaaga and aagaa, that occurred with the highest frequency in proximity to intron-exon and exon-intron boundaries. go analyses based on these interactions suggested safb was likely to have roles in regulating expression of proteins involved in rna processing, the cellular response to stress, and neuron projection and morphogenesis. ncam synpo astn and pde4b are examples of genes with multiple safb tags and further investigation, including ncam minigene experiments, confirmed safb knockdown did alter their expression. interestingly, ncam, astn, and pde4b are known to play important roles in regulating synaptic function and are implicated in human psychiatric disease. importantly, safb was also found to regulate the processing of non-coding rnas. safb has multiple cross-link sites along the lncrna malat and safb knockdown increased malat expression. malat has been shown to interact with multiple sr proteins to regulate alternative splicing. previous studies using iclip have shown that the splice regulators tdp- srsf, srsf and srsf also cross-link extensively with malat and this interaction may therefore be common with rbps that regulate splicing. iclip-seq analyses also showed that safb interacted with the physiologically important mir-17- mirna cluster. we further showed that this interaction had functional consequences, with safb knockdown being found to significantly increase the expression of the mir-17- primary transcript and reduce the expression of mir-19a. interestingly, and related to the functions predicted by the go analyses, the mir-17- cluster has been shown to be an important regulator of axonal function. furthermore, mir-17- cluster dysregulation has been implicated in a number of cancers, human aging and in neurodegenerative disease. in this study, we validated interactions with genes that had a high and comparatively low number of safb tags suggesting the presence of iclip tags was a good predictor of functionally important interactions. to further investigate the role safb plays in alternative splicing we used rnamotifs to analyse the enrichment and distribution of the gaa, aag and aga binding motifs in differentially expressed exons. this analysis allowed position-dependent effects on exon inclusion/exclusion following safb binding to be predicted. the frequency of one or more motif pairs was significantly increased within downregulated exons suggesting safb in these cases promoted exon inclusion. safb iclip tags were found in the ncam gene and ncam minigene experiments showed that safb knockdown altered alternative splicing and that mutation of the gaa, aag and aga motifs significantly decreased this regulation. these data therefore support the iclip and rnamotifs analyses, which together suggested safb regulates the alternative splicing of specific genes via recognition of ag rich motifs. safb has been implicated in the regulation of a number of cellular processes; however, as it is a multi-domain protein defining specific roles has proved difficult. the regulation of ncam splicing and its interaction with known splicing regulators and with polymerase ii suggests it may be involved in spliceosome assembly. we provide evidence supporting this hypothesis and suggest that safb may act to recruit splicing factors to a small number of pre-mrnas. the dead-box helicase eif4aiii, a core component of the exon junction complex was found to bind within exon regions rich in gaaga motifs at sites distal and close to ejc regions. as well as safb and eif4aiii, a number of sr proteins have been found to bind the n motif, including srsf, htra and srm. it was suggested that gaaga might be a high-affinity binding site for factors that influence ejc binding; as such, it is possible that these proteins bind this motif to attract ejc factors during splicing and/or to stabilize the ejc in specific genes. safb expression levels differ between tissues and, as its expression levels in neurons are high, it is therefore possible that safb plays a more significant role in regulating rna processing in these tissues. the level and sub-cellular location of safb also differs following stress and this may also influence the degree to which safb competes with other proteins for the recognition site. similarly, as safb is known to bind other splice regulators, its expression levels may also influence the sequence preference/specificity of spliceosome complexes and thereby regulate cellular function. go functional analyses suggested safb regulates the expression of proteins involved in atp/nucleoside binding, cytoskeletal organisation and microtubule-based processes. importantly, minigene experiments showed the alternative splicing of ncam was altered by safb ncam is an important regulator of synaptic function and its altered expression and splicing are implicated in the aetiology of human neuropsychiatric illnesses. safb mediated isoform-specific changes in ank, anks1b, sap, add, kif16b, and elk, as well as in genes linked to the cytoskeleton and/or synaptic function and the aetiology of neurologic disease. for example, the ankyrin-g protein encoded by ank organizes synaptic microtubules and is involved in protein trafficking, neurogenesis and neuroprotection, and in the aetiology of bipolar disorder and schizophrenia. safb is located at 19p -p and separate studies have found snps or duplications at this locus are associated with increased susceptibility to schizophrenia. these data suggest that altered function of safb could result in changes in the expression of genes that regulate synaptic function, some of which are also susceptibility genes for schizophrenia.  our global analysis shows safb interacts with distinct classes of rna indicating it has multiple functions with regard to rna processing and the control of gene expression and function. importantly, we confirmed it binds primarily within exons and that safb1’s altered expression regulates the alternative splicing of genes with important roles in neuronal function. iclip data and further analysis using rnamotifs predicted that safb is likely to interact with specific purine-rich trimer sequences to promote exon inclusion. the safb family consists of safb safb and sltm, proteins that share considerable identity and are all highly but differentially expressed in the central nervous system. the safb proteins will therefore have novel and unexplored roles in regulating the processing of coding and non-coding pre-mrnas in the brain and, as such, represent a new and important family of rna regulators. 
together, our data suggest safb will play an important physiological role in the central nervous system regulating synaptic function. these genes regulate splicing, axonal and synaptic function, and are associated with neuropsychiatric disease, suggesting that safb is an important regulator of key neuronal processes. safb is a rna binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, dna repair and rna processing. we found that safb regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion. to further investigate the association of safb with splicing we used exon array analysis and found safb knockdown mediated the statistically significant up- and downregulation of alternative exons. further analysis using rnamotifs to investigate the frequency of association between the motif pairs and alternative spliced exons found there was a highly significant correlation with downregulated exons. iclip analysis found safb binding was enriched, specifically in exons, ncrnas, 3’ and 5’ untranslated regions.
 safb is a rna binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, dna repair and rna processing. to gain further insight into safb function we used iclip and mapped its interaction with rna on a genome wide level.  iclip analysis found safb binding was enriched, specifically in exons, ncrnas, 3’ and 5’ untranslated regions. safb was found to recognise a purine-rich gaaga motif with the highest frequency and it is therefore likely to bind core aga, gaa, or aag motifs. confirmatory rt-pcr experiments showed that the expression of coding and non-coding genes with safb cross-link sites was altered by safb knockdown. for example, we found that the isoform-specific expression of neural cell adhesion molecule and astn was influenced by safb and that the processing of mir-19a from the mir-17- cluster was regulated by safb these data suggest safb may influence alternative splicing and, using an ncam minigene, we showed that safb knockdown altered the expression of two of the three ncam alternative spliced isoforms. however, when the aga, gaa, and aag motifs were mutated, safb knockdown no longer mediated a decrease in the ncam 9– alternative spliced form. to further investigate the association of safb with splicing we used exon array analysis and found safb knockdown mediated the statistically significant up- and downregulation of alternative exons. further analysis using rnamotifs to investigate the frequency of association between the motif pairs and alternative spliced exons found there was a highly significant correlation with downregulated exons. together, our data suggest safb will play an important physiological role in the central nervous system regulating synaptic function. we found that safb regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion.  iclip showed that safb has previously uncharacterised specific rna binding properties that help coordinate the isoform-specific expression of coding and non-coding genes. these genes regulate splicing, axonal and synaptic function, and are associated with neuropsychiatric disease, suggesting that safb is an important regulator of key neuronal processes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hnrnpicliplong non-coding rnamirnancam1neuronalrnasafb1splicinghttp://dx.doi.org/ /501100000268biotechnology and biological sciences research councilj0164891/1bb/f0222981/1http://dx.doi.org/ /100004440wellcome trust089583issue-copyright-statement© the author 2015 