Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Genes Dev. 2015 Oct 15;29(20):2168-82. doi: 10.1101/gad.270421.115. Epub 2015 Oct 8.


Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes.

Keywords: Laccase2; PlexA; circRNA; hnRNP; noncoding RNA; pre-mRNA splicing; repetitive element.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Pairing
  • Drosophila Proteins / genetics
  • Drosophila melanogaster / genetics*
  • Gene Expression Regulation, Enzymologic*
  • Heterogeneous-Nuclear Ribonucleoproteins / genetics*
  • Humans
  • Introns / genetics*
  • Laccase / biosynthesis*
  • Laccase / genetics*
  • Microsatellite Repeats / genetics
  • Nerve Tissue Proteins / genetics
  • RNA / genetics*
  • Receptors, Cell Surface / genetics
  • Serine-Arginine Splicing Factors / genetics


  • Drosophila Proteins
  • Heterogeneous-Nuclear Ribonucleoproteins
  • Nerve Tissue Proteins
  • Receptors, Cell Surface
  • plexA protein, Drosophila
  • Serine-Arginine Splicing Factors
  • RNA
  • Laccase