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, basepairing 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.

Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

“…Strikingly, many alternatively spliced cassette exons appeared to be circRNApredominant. Although the detailed mechanism is unclear, it is possible that such events could occur post-transcriptionally during circRNA biogenesis (Kramer et al 2015;Zhang et al 2016). Finally, the biological significance of circRNA-predominant alternative splicing awaits further investigation.…”

Circular RNAs: Unexpected outputs of many protein-coding genes

“…For example, circular RNAs may allow the formation of large RNA-protein complexes, e.g., at neuronal synapses, 32,33 or possibly be translated. [34][35][36][37] This review highlights recent advances that help explain how the choice between linear vs. circular RNA production is made. Many circular RNAs are expressed in a tissue-specific manner and at low levels, 29,[31][32][33]38 likely because backsplicing is far less efficient than canonical splicing.…”

“…When exons are flanked by long complementary repeats that efficiently bring the splice sites into close proximity (see below), backsplicing can occur more rapidly and premRNA 3 0 end processing is not required. 37 How then does the Figure 1. A pre-mRNA can be spliced to generate a linear or circular RNA.…”

“…A pair of DNAREP1_DM family transposons are close to the circularizing exon and facilitate backsplicing. 37 Although very few exons are flanked by repeats as long as those at the mouse Sry locus, most (>70 %) 39 exons that generate circular RNAs in human cells have complementary repeats, usually Alu elements, in their flanking introns. 14,40,68 Circular RNAs in other species, including mice, C. elegans, 68 and Drosophila, 37 are often also flanked by complementary intronic repeats (but not in all cases 25,29 ; see below).…”

“…40 RNA binding proteins further regulate circular RNA biogenesis in a combinatorial manner, allowing tight control over which mature transcripts are generated. 37 At certain genes, circular RNAs are produced independently of repetitive elements and the backsplicing reaction is instead coupled to an exon skipping event. 41 In addition, a separate class of circular intronic RNAs are made not by backsplicing but by a failure to debranch intron lariats.…”