1. The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential
- Author
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Cristina Barbagallo, Michele Stella, Cinzia Di Pietro, Roberto Altieri, Thomas B. Hansen, Angela Caponnetto, Gabriella Lupo, Davide Barbagallo, Rosalia Battaglia, Duilia Brex, Giuseppe Barbagallo, Marco Ragusa, Giuseppe Broggi, Francesco Certo, Michele Purrello, Carmelina Daniela Anfuso, Rosario Caltabiano, and Federica Mirabella
- Subjects
Angiogenesis ,Chromosomal Proteins, Non-Histone ,RNA-binding protein ,Apoptosis ,RNA-binding proteins ,lcsh:Chemistry ,angiogenesis ,Cell Movement ,circSMARCA5 ,Tumor Cells, Cultured ,lcsh:QH301-705.5 ,Spectroscopy ,Tube formation ,Adenosine Triphosphatases ,Neovascularization, Pathologic ,Serine-Arginine Splicing Factors ,Chemistry ,General Medicine ,Transfection ,Prognosis ,CircSMARCA5 ,Computer Science Applications ,Gene Expression Regulation, Neoplastic ,SRSF1 ,Vascular endothelial growth factor A ,SRSF3 ,VEGFA ,Binding site mutation ,binding site mutation ,Catalysis ,Article ,Inorganic Chemistry ,An-giogenesis ,Biomarkers, Tumor ,Humans ,Physical and Theoretical Chemistry ,Binding site ,Nucleotide Motifs ,Molecular Biology ,Cell Proliferation ,Messenger RNA ,Organic Chemistry ,RNA ,Endothelial Cells ,RNA, Circular ,Molecular biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Glioblastoma - Abstract
Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.
- Published
- 2020
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