Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells

PLoS One. 2016 Oct 13;11(10):e0164085. doi: 10.1371/journal.pone.0164085. eCollection 2016.

Abstract

Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology.

MeSH terms

  • Alternative Splicing*
  • Exons
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Polymerase Chain Reaction
  • RNA / genetics*
  • RNA Precursors / genetics
  • RNA, Circular
  • Repressor Proteins / genetics*
  • Single-Cell Analysis

Substances

  • ASXL1 protein, human
  • RNA Precursors
  • RNA, Circular
  • Repressor Proteins
  • RNA

Grant support

This work was supported by funding to CG from the Burroughs Wellcome Fund (http://www.bwfund.org/grant-programs/biomedical-sciences/career-awards-medical-scientists), Leukemia and Lymphoma Society (http://www.lls.org), American Society of Hematology (http://www.hematology.org/Awards/Career-Training/463.aspx), and American Lebanese Syrian Associated Charities (Fundraiser for St. Jude Children's Hospital—https://www.stjude.org/about-st-jude/leadership/alsac-leadership.html). WK was also supported by A*STAR, agency of science, technology and research, Singapore (https://www.a-star.edu.sg). Some of these studies were supported by the Howard Hughes Medical Institute (http://www.hhmi.org) by funding from POB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.