The paralogues MAGOH and MAGOHB are oncogenic factors in high-grade gliomas and safeguard the splicing of cell division and cell cycle genes

Abstract

The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.

Keywords: RNA-binding proteins; cancer genomics; gene expression regulation; glioblastoma; glioma; splicing.

Figure 1.

MAGOH and MAGOHB are overexpressed in multiple cancer types. A) MAGOH, MAGOHB, and MAGOH plus MAGOHB expression in multiple normal tissues from the GTEx database; Spearman correlation coefficients (rho) are shown between MAGOH and MAGOHB. B) MAGOH and MAGOHB genomic locations (top) and protein sequence alignment (bottom). Untranslated regions are shown in gray. C) MAGOH and MAGOHB expression (DESeq2 normalized) during cortex development according to Cortecon [36]. D) Combined MAGOH/MAGOHB expression levels in TCGA tumor samples vs. corresponding normal tissues from the GTEx database. Gray bars indicate the fold change between tumor versus normal expression (median). THCA, thyroid carcinoma; PRAD, prostate adenocarcinoma; ESCA, esophageal carcinoma; LGG, brain lower grade glioma; LUAD, lung adenocarcinoma; KIRC, kidney renal clear cell carcinoma; UCEC, uterine corpus endometrial carcinoma; BRCA, breast invasive carcinoma; LIHC, liver hepatocellular carcinoma; STAD, stomach adenocarcinoma; COAD, colon adenocarcinoma; PAAD, pancreatic adenocarcinoma; BLCA, bladder urothelial carcinoma; HGG, high grade (grade 4) glioma. Distributions of MAGOH/MAGOHB expression in tumor versus normal tissues were compared with Wilcoxon rank-sum tests. P-value ≤ 0.01 (**) and p-value ≤ 0.0001 (****).

Figure 2.

Levels of MAGOH and MAGOHB expression correlate with degree of malignancy and predict outcomes in glioma patients. A) Expression of MAGOH/MAGOHB in the brain cortex for grades 2, 3, and 4 gliomas (****: p-value < 0.0001; Wilcoxon rank-sum test). B) Immunohistochemical staining for MAGOH and MAGOHB in glioma samples from the Shanghai ChangZheng Hospital cohort. The antibody (ab170944) detects both MAGOH and MAGOHB proteins as they are almost identical (98.63%), differing only by the first two amino-acids, Figure 1B. C) Survival curves of glioma patients with high (blue) and low (red) combined MAGOH and MAGOHB expression in three independent cohorts.

Figure 3.

MAGOH and MAGOHB knockdown impacts cancer-relevant phenotypes. A) Cell viability was determined by MTS assays. MAGOH/MAGOHB knockdown significantly reduced viability of U251 and U343 glioblastoma cells and 1919 and 3565 glioma stem cells (**p-value <0.01, *p-value < 0.05; both, Student’s T-test). No changes were observed in astrocytes (AST). B) Proliferation was reduced in U251 and U343 MAGOH/MAGOHB knockdown cells compared to siControl cells; measured on an IncuCyte^→ system (***p-value <0.001, ANOVA). C) Caspase-3/7 activity indicated increased apoptosis in U251 and U343 MAGOH/MAGOHB knockdown cells versus siControl cells. Astrocytes (AST) showed no changes in caspase activity (**p-value <0.01, T-test). D) Reduced MAGOH/MAGOHB expression caused changes in cell cycle distribution with G1 arrest in U251 and U343 cells (***p-value <0.001, t-test).

Figure 4.

MAGOH/MAGOHB knockdown alters the splicing profile of GBM cells. (A) Splicing events showing changes after MAGOH/MAGOHB knockdown in GBM cell lines (|ΔPSI| > 0.2 and false discovery rate [FDR] < 0.01). (B) Altered splicing events in U251 and U343 cells stratified by event type: skipped exons (SE), retained introns (RI), mutually exclusive exons (MXE), and alternative 5′ and 3′ splice sites (A5SS and A3SS). (C) Enriched GO terms (biological processes) for genes displaying splicing alterations upon MAGOH/MAGOHB KD in both U251 and U343 cells (hypergeometric test; FDR < 0.05). (D) Protein-protein interaction networks of genes displaying the same splicing alteration in MAGOH/MAGOHB KD cells in both GBM cell lines. Node genes are shown in gray.

Figure 5.

MAGOH/MAGOHB knockdown promotes multiple exon- skipping and aberrant splicing events. A) Model displaying how the EJC can prevent recursive splicing. After splicing, a new splice site can originate in the newly formed exon junction. EJC occupancy near this new junction can prevent a new splicing event (recursive splicing). B) EJC occupancy is elevated in the exon upstream to the skipped exon(s) regulated by MAGOH/MAGOHB compared to other exons in the same gene (excluding the last exon). Bootstrap curves (100,000 re-samplings) of random selected exons indicate that exons immediately upstream to skipped exons (red line) have a higher ratio of EJC occupancy than the remaining exons. Values obtained for the further upstream exons (blue line) are similar to those of other exons. C) MAGOH/MAGOHB knockdown cells had more RNA-seq reads (reflecting skipping of one or more exons) compared to controls. D) Model proposing that exons with high EJC occupancy are more sensitive to MAGOH/MAGOHB KD, resulting in multiple exon-skipping events. E) MAGOH/MAGOHB knockdown cells display a higher ratio (median) of reads, supporting skipping of two or more exons (e.g. 2x and 3x for skipping 2 and 5 exons, respectively).

Figure 6.

Genes affected by MAGOH/MAGOHB knockdown presenting with multiple exon-skipping events are mainly associated with regulation of cell cycle/division. (A) Genes with multiple exon-skipping events in both U251 and U343 MAGOH/MAGOHB KD cells and their functions. (B) Cell cycle and cell division genes whose protein domains were compromised (partial or total domain losses) due to multiple exon-skipping events in MAGOH/MAGOHB KD cells. (C) Genes presenting partial or total domain losses due to multiple exon-skipping events caused by MAGOH/MAGOHB KD are implicated in different phases of the cell cycle.