Exon circularization requires canonical splice signals

Cell Rep. 2015 Jan 6;10(1):103-11. doi: 10.1016/j.celrep.2014.12.002. Epub 2014 Dec 24.


Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics*
  • Exons / genetics*
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Introns / genetics
  • Nucleic Acid Conformation
  • Protein Serine-Threonine Kinases / genetics*
  • RNA Precursors / genetics
  • RNA Splice Sites / genetics*
  • RNA, Untranslated / genetics*
  • Spliceosomes / genetics


  • Intracellular Signaling Peptides and Proteins
  • RNA Precursors
  • RNA Splice Sites
  • RNA, Untranslated
  • HIPK3 protein, human
  • Protein Serine-Threonine Kinases