Short intronic repeat sequences facilitate circular RNA production

Genes Dev. 2014 Oct 15;28(20):2233-47. doi: 10.1101/gad.251926.114. Epub 2014 Oct 3.


Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.

Keywords: Alu; EPHB4; HIPK3; ZKSCAN1; circRNA; noncoding RNA; splicing.

Publication types

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

MeSH terms

  • Base Pairing
  • Base Sequence
  • Humans
  • Inteins / genetics*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Microsatellite Repeats / genetics*
  • Molecular Sequence Data
  • Mutation
  • Plasmids / genetics
  • Protein Serine-Threonine Kinases / genetics
  • RNA / biosynthesis*
  • RNA / genetics*
  • RNA Splice Sites / genetics
  • RNA, Circular
  • Transcription Factors / genetics


  • Intracellular Signaling Peptides and Proteins
  • RNA Splice Sites
  • RNA, Circular
  • Transcription Factors
  • RNA
  • HIPK3 protein, human
  • Protein Serine-Threonine Kinases