title: Alternative splicing factor or splicing factor-2 plays a key role in intron retention of the endoglin gene during endothelial senescence.
abstract: Alternative splicing involving intron retention plays a key role in the regulation of gene expression. We previously reported that the alternatively spliced short isoform of endoglin (S-endoglin) is induced during the aging or senescence of endothelial cells by a mechanism of intron retention. In this work, we demonstrate that the alternative splicing factor or splicing factor-2 (ASF/SF2) is involved in the synthesis of endoglin. Overexpression of ASF/SF2 in endothelial cells switched the balance between the two endoglin isoforms, favoring the synthesis of S-endoglin. Using a minigene reporter vector and RNA immunoprecipitation experiments, it was shown that ASF/SF2 interacts with the nucleotide sequence of the endoglin minigene, suggesting the direct involvement of ASF/SF2. Accordingly, the sequence recognized by ASF/SF2 in the endoglin gene was identified inside the retained intron near the consensus branch point. Finally, the ASF/SF2 subcellular localization during endothelial senescence showed a preferential scattered distribution throughout the cytoplasm, where it interferes with the activity of the minor spliceosome, leading to an increased expression of S-endoglin mRNA. In summary, we report for the first time the molecular mechanisms by which ASF/SF2 regulates the alternative splicing of endoglin in senescent endothelial cells, as well as the involvement of ASF/SF2 in the minor spliceosome.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, and Centro de Investigación Biomédica en Red de Enfermedades Raras, c/Ramiro de Maeztu 9, Madrid, Spain. fjblanco@cib.csic.es
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.forename: Carmelo
authors.initials: C
authors.lastname: Bernabeu
mesh_terms: D017398:Alternative Splicing; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D016922:Cellular Senescence; D017124:Conserved Sequence; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D000071063:Endoglin; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007438:Introns; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D000068103:Serine-Arginine Splicing Factors