CircSMARCA5 Inhibits Migration of Glioblastoma Multiforme Cells by Regulating a Molecular Axis Involving Splicing Factors SRSF1/SRSF3/PTB.

Davide Barbagallo,Angela Caponnetto,Rosario Caltabiano,Thomas Hansen,Cinzia Di Pietro,Matilde Cirnigliaro,Antonio Morrone,Marco Ragusa,Cristina Barbagallo,Giuseppe Barbagallo,Michele Purrello,Floriana D’Angeli,Duilia Brex

doi:10.3390/ijms19020480

Abstract

Circular RNAs (circRNAs) have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM) pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5): it was significantly downregulated in GBM biopsies as compared to normal brain tissues (p-value < 0.00001, student’s t-test), contrary to its linear isoform counterpart that did not show any differential expression (p-value = 0.694, student’s t-test). Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma’s histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs), specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1), a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE). Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3) RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD) substrate. Interestingly, SRSF3 is known to interplay with two other splicing factors, polypyrimidine tract binding protein 1 (PTBP1) and polypyrimidine tract binding protein 2 (PTBP2), that positively regulate glioma cells migration. Collectively, our data show circSMARCA5 as a promising druggable tumor suppressor in GBM and suggest that it may exert its function by tethering the RBP SRSF1.

Highlights

Circular RNAs have recently been established as a comprehensive class of RNAs generated by non-linear back-splicing [1,2,3,4,5,6]
This hypothesis is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data from Encyclopedia of DNA Elements (ENCODE) (Figure 4) as well as by the circSMARCA5-mediated changes in serine and arginine rich splicing factor 3 (SRSF3) splicing pattern (Figure 5A) and by data on glioblastoma multiforme (GBM) biopsies (Figure 5B,C)
These data support our hypotheses that circSMARCA5 may indirectly regulate the expression of SRSF3 isoforms, by tethering serine and arginine rich splicing factor 1 (SRSF1), and, in turn, functional SRSF3 protein may positively regulate the expression of SRSF3 Ex4, which is normally expressed at very low levels within cells (Figure S6)

Summary

Introduction

Circular RNAs (circRNAs) have recently been established as a comprehensive class of RNAs generated by non-linear back-splicing [1,2,3,4,5,6]. Most studies describe circRNAs as microRNA (miRNA) sponges, for some of them, a role as modulators of RNA binding proteins (RBPs) has been suggested [21,22,23,24,25,26,27,28]. We focused on circSMARCA5, the most downregulated circRNA (among the twelve analyzed) in GBM samples as compared to healthy brain parenchyma, and characterized it as a novel tumor-suppressor, regulating the migration of GBM cells. Our data indicate that circSMARCA5 may exert its function by modulating the RNA binding protein serine and arginine rich splicing factor 1 (SRSF1), a known oncoprotein involved in positive regulation of cell migration [29]

Methods

Results

Conclusion