Fig 1: Serine/arginine repetitive matrix 4 (SRRM4) is a bona fide circRNA and ME-circRNA biogenesis factor. (a) qRT-PCR comparing relative expression of SRRM transcripts between healthy brain tissue (H) and GBM tumor tissue (T). Normalized to GAPDH, n = 4–5 tissue samples. One-way ANOVA, Mann–Whitney test was performed to assess significance. n.s.: no significant difference (p > 0.05) (b) Western blot using FLAG antibody on cell protein lysates from HEK293 cells transiently transfected with pcDNA3.1 (EV), pcDNA3.1::SRRM3-FLAG (SRRM3) or pcDNA3.1::SRRM4-FLAG (SRRM4). Bands of expected size for SRRM3 (70 kDa) and SRRM4 (65 kDa). (c) RT-PCR for ACVR2A linear (ACVR2A), and SRRM4-specific ACVR2A RNA with ME, circACVR2A (comprising exons 3-8) and increase in MEF2C linear RNA with ME. (d) RT-PCR on 3 biological replicates of HEK293 cells with EV, or SRRM4 overexpression for SRRM4, REST (2 bands, with upper band representing the inclusion of ME in REST4), ACVR2A (ME) and GAPDH used as loading control. (e) Scaled Venn diagram showing total number of circRNAs identified across the three replicate EV and SRRM4 overexpression circRNA-seq libraries using three different circRNA identification pipelines, CIRCexplorer2, CIRI and Hyb. (f) RT-PCR for two novel SRRM4-driven circRNAs showing absence in HEK293 EV cells. (g) Volcano plot comparing circRNA expression between EV and SRRM4 overexpression. (h) Scaled Venn diagram showing ME-circRNAs, with the ME present at the backsplice junction, identified using Hyb. (i) RT-PCR showing the dependence on SRRM4 for expression of ME-circEIF4G3 from transgenic circRNA overexpression construct. Top, HeLa cells (with high levels of SRRM4 expression) could produce ME-circEIF4G3 with and without SRRM4 overexpression 24 h post-transfection. Bottom, HEK293 cells (lacking SRRM4 expression) could only produce the circRNA if SRRM4 was co-transfected. * are non-specific RT-PCR products.