title: Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Marianne C
authors.initials: MC
authors.lastname: Kramer
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Dongming
authors.initials: D
authors.lastname: Liang
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Deirdre C
authors.initials: DC
authors.lastname: Tatomer
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Gold
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: March
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Cherry
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Jeremy E
authors.initials: JE
authors.lastname: Wilusz
mesh_terms: D000818:Animals; D020029:Base Pairing; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D042845:Laccase / Q000096:biosynthesis* / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics; D000068103:Serine-Arginine Splicing Factors / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; C116483:plexA protein, Drosophila; D000068103:Serine-Arginine Splicing Factors; D012313:RNA; D042845:Laccase
keywords: Laccase2; PlexA; circRNA; hnRNP; noncoding RNA; pre-mRNA splicing; repetitive element