Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes

Abstract

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A–2B–3–4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A–2B–3–4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A–2B–3–4. About half of the affected genes by C2A–2B–3–4 remained unaffected by L2A–2B–3–4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A–2B–3–4 in gene regulation. A surprisingly high number of upregulated genes by C2A–2B–3–4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A–2B–3–4 and L2A–2B–3–4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A–2B–3–4 with very limited overlap with those affected by L2A–2B–3–4. Independent validations confirmed the effect of C2A–2B–3–4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A–2B–3–4, and expands our understanding of functions of SMN1/2 genes.