Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells.
Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3' or 5' untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells.
Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Elaine
E
Hong
Institute of Genetic Medicine, Faculty of Medical Sciences, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK.
Andrew
A
Best
Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Hannah
H
Gautrey
Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Jas
J
Chin
Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Anshuli
A
Razdan
Faculty of Computer and Information Science, University of Ljubljana, Traska Cesta 25, 51-1000 Ljubljana, Slovenia.
Tomaz
T
Curk
Institute of Genetic Medicine, Faculty of Medical Sciences, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK.
David J
DJ
Elliott
Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Alison J
AJ
Tyson-Capper
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D004967:Estrogens / Q000235:genetics; D005260:Female; D006801:Humans; D007438:Introns / Q000235:genetics; D061986:MCF-7 Cells; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics*; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D012313:RNA / Q000235:genetics*; D034622:RNA Interference / Q000502:physiology; D016601:RNA-Binding Proteins / Q000235:genetics*; D011960:Receptors, Estrogen / Q000235:genetics*
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D004967:Estrogens; D036961:Matrix Attachment Region Binding Proteins; D034521:Nuclear Matrix-Associated Proteins; D016601:RNA-Binding Proteins; D011960:Receptors, Estrogen; C109132:SAFB protein, human; C486369:SAFB2 protein, human; D012313:RNA