title: iCLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function.
abstract: BACKGROUND
  SAFB1 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 SAFB1 function we used iCLIP and mapped its interaction with RNA on a genome wide level.
  
  
  RESULTS
  iCLIP analysis found SAFB1 binding was enriched, specifically in exons, ncRNAs, 3' and 5' untranslated regions. SAFB1 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 SAFB1 cross-link sites was altered by SAFB1 knockdown. For example, we found that the isoform-specific expression of neural cell adhesion molecule (NCAM1) and ASTN2 was influenced by SAFB1 and that the processing of miR-19a from the miR-17-92 cluster was regulated by SAFB1. These data suggest SAFB1 may influence alternative splicing and, using an NCAM1 minigene, we showed that SAFB1 knockdown altered the expression of two of the three NCAM1 alternative spliced isoforms. However, when the AGA, GAA, and AAG motifs were mutated, SAFB1 knockdown no longer mediated a decrease in the NCAM1 9-10 alternative spliced form. To further investigate the association of SAFB1 with splicing we used exon array analysis and found SAFB1 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 (AGA followed by AGA, GAA or AAG) and alternative spliced exons found there was a highly significant correlation with downregulated exons. Together, our data suggest SAFB1 will play an important physiological role in the central nervous system regulating synaptic function. We found that SAFB1 regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion.
  
  
  CONCLUSIONS
  iCLIP showed that SAFB1 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 SAFB1 is an important regulator of key neuronal processes.
authors.affiliation: Regenerative Medicine Laboratories, School of Clinical Sciences, Cellular & Molecular Medicine, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK. mdcar@bristol.ac.uk.
authors.forename: Caroline
authors.initials: C
authors.lastname: Rivers
authors.affiliation: Regenerative Medicine Laboratories, School of Clinical Sciences, Cellular & Molecular Medicine, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK. ji1569@bristol.ac.uk.|Institute of Medical Sciences & Technology, University of Kuala Lumpur, Kuala Lumpur, 43000, Malaysia. ji1569@bristol.ac.uk.
authors.forename: Jalilah
authors.initials: J
authors.lastname: Idris
authors.affiliation: Regenerative Medicine Laboratories, School of Clinical Sciences, Cellular & Molecular Medicine, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK. anhls@bristol.ac.uk.
authors.forename: Helen
authors.initials: H
authors.lastname: Scott
authors.affiliation: Intelligent Systems Laboratory, Department of Engineering & Mathematics, Merchant Venturers Building, University of Bristol, Bristol, BS8 1UB, UK. mark.rogers@bristol.ac.uk.
authors.forename: Mark
authors.initials: M
authors.lastname: Rogers
authors.affiliation: MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London, UK. younbok.lee@kcl.ac.uk.
authors.forename: Youn-Bok
authors.initials: YB
authors.lastname: Lee
authors.affiliation: Regenerative Medicine Laboratories, School of Clinical Sciences, Cellular & Molecular Medicine, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK. jessica.gaunt@bristol.ac.uk.
authors.forename: Jessica
authors.initials: J
authors.lastname: Gaunt
authors.affiliation: Faculty of Computer and Information Science, University of Ljubljana, Trzaska cesta 25, SI-1001, Ljubljana, Slovenia. laphylac@cing.ac.cy.
authors.forename: Leonidas
authors.initials: L
authors.lastname: Phylactou
authors.affiliation: The Cyprus Institute of Neurology & Genetics, PO Box 23462, 1683, Nicosia, Cyprus. tomaz.curk@fri.uni-lj.si.
authors.forename: Tomaz
authors.initials: T
authors.lastname: Curk
authors.affiliation: Institute of Medical Sciences & Technology, University of Kuala Lumpur, Kuala Lumpur, 43000, Malaysia. campbell@bristol.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Campbell
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK. j.ule@ucl.ac.uk.
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.affiliation: Regenerative Medicine Laboratories, School of Clinical Sciences, Cellular & Molecular Medicine, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK. m.r.norman@bristol.ac.uk.
authors.forename: Michael
authors.initials: M
authors.lastname: Norman
authors.affiliation: Regenerative Medicine Laboratories, School of Clinical Sciences, Cellular & Molecular Medicine, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK. james.uney@bristol.ac.uk.
authors.forename: James B
authors.initials: JB
authors.lastname: Uney
mesh_terms: D017398:Alternative Splicing; D019002:CD56 Antigen / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C000593289:ASTN2 protein, human; D019002:CD56 Antigen; D006023:Glycoproteins; D036961:Matrix Attachment Region Binding Proteins; C522918:NCAM1 protein, human; D009419:Nerve Tissue Proteins; D034521:Nuclear Matrix-Associated Proteins; D022661:RNA, Untranslated; D011960:Receptors, Estrogen; C109132:SAFB protein, human