title: Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells.
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.
authors.affiliation: Departments of Bioengineering and Applied Physics, Stanford University, Stanford, CA, 94305, United States of America.
authors.forename: Winston
authors.identifier: http://orcid.org/0000-0003-4611-6490
authors.initials: W
authors.lastname: Koh
authors.affiliation: Departments of Oncology and Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, United States of America.
authors.forename: Veronica
authors.identifier: http://orcid.org/0000-0002-0645-7365
authors.initials: V
authors.lastname: Gonzalez
authors.affiliation: Departments of Oncology and Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, United States of America.
authors.forename: Sivaraman
authors.initials: S
authors.lastname: Natarajan
authors.affiliation: Departments of Oncology and Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Carter
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Stanford University, Stanford, CA, 94305, United States of America.
authors.forename: Patrick O
authors.initials: PO
authors.lastname: Brown
authors.affiliation: Departments of Oncology and Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, United States of America.
authors.forename: Charles
authors.identifier: http://orcid.org/0000-0002-4728-8203
authors.initials: C
authors.lastname: Gawad
mesh_terms: D017398:Alternative Splicing; D005091:Exons; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics; D000079962:RNA, Circular; D012097:Repressor Proteins / Q000235:genetics*; D059010:Single-Cell Analysis
chemical_list: C473949:ASXL1 protein, human; D012322:RNA Precursors; D000079962:RNA, Circular; D012097:Repressor Proteins; D012313:RNA