The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential

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.

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein, Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor

“…To understand the NRF1 and SMAD4 protein-protein interaction more completely, an extensive analysis of each protein and/or structural biology studies will be required. Recently, Barbagallo, D et al and Broggi, G et al reported that the RNA-binding motif responsible for the interaction between Serine and Arginine Rich Splicing Factor 1 (SRSF1) and circSMARCCA5 (a 269-nucleotide-long circRNA) are coupled with increased amounts of total Vascular Endothelial Growth Factor A (VEGFA) mRNA secretion and the recommended use of SRSF1 as a diagnostic immunomarker in gliomas [34,35]. In the case of NRF1, it appears to be both a negative regulator and an interaction partner of SMAD4.…”

Diagnostic Utility of the Immunohistochemical Expression of Serine and Arginine Rich Splicing Factor 1 (SRSF1) in the Differential Diagnosis of Adult Gliomas

“…Cell migration was assayed through Oris™ Cell Migration Assay (Platypus Technologies, Madison, WI, USA), as previously described [ 27 ]. Briefly, 3.5 × 10 4 transfected cells/well were seeded in a 96-well migration plate with the stoppers placed.…”

“…( C ) Bar graph showing the mean ± standard deviation of the percentage (%) of NC or SRSF1-transfected U87MG cell migration. The percentage of cell migration has been calculated as previously reported [ 27 ]. * p -value < 0.05 ( n = 6, Student’s t -test vs. control).…”

Pro- and Antiangiogenic Factors in Gliomas: Implications for Novel Therapeutic Possibilities

“…Circular RNAs may be involved in the regulation of glioma cell proliferation, migration, and invasion. They may be also involved in glioma angiogenesis via miRNA-526b-3p/MMP2 pathway and RNA-binding proteins [ 254 , 255 ]. Circ-ATXN1 were significantly upregulated in glioma-associated endothelial cells in comparison with astrocyte-associated endothelial cells (AECs), whereas knockdown of circ-ATXN1 significantly inhibited endothelial cell viability, migration and tube formation [ 255 ].…”