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

## TL;DR

This study shows that a circular RNA from SMA genes affects thousands of genes and proteins, highlighting its unique regulatory role.

## Contribution

The study reveals the transcriptome- and proteome-wide effects of a specific circular RNA from SMA genes.

## Key findings

- Overexpression of C2A-2B-3–4 altered ~15% of genes, with limited overlap with a linear transcript.
- C2A-2B-3–4 upregulated genes on chromosomes 4 and 7 and downregulated genes on chromosomes 10 and X.
- C2A-2B-3–4 affected proteins related to chromatin, transcription, and neuromuscular junction formation.

## Abstract

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2, produce multiple circular RNAs (circRNAs), including C2A-2B-3–4 that encompasses early exons 2A, 2B, 3 and 4. 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 (4,172 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/SMN2. These fifindings 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/SMN2 transcripts, C2A-2B-3–4 and L2A-2B-3–4 upregulated and downregulated SMN1/SMN2 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, a universally expressed circRNA produced by SMN1/SMN2.