Molecular and genetic dissection of recursive splicing

Intronic ratchet points (RPs) are abundant within long introns in the Drosophila genome and consist of juxtaposed splice acceptor and splice donor (SD) sites. Although they appear to encompass zero-nucleotide exons, we recently clarified that intronic recursive splicing (RS) requires a cryptic exon at the RP (an RS-exon), which is subsequently always skipped and thus absent from mRNA. In addition, Drosophila encodes a smaller set of expressed exons bearing features of RS. Here, we investigate mechanisms that regulate the choice between RP and RS-exon SDs. First, analysis of Drosophila RP SD mutants demonstrates that SD competition suppresses inclusion of cryptic exons in endogenous contexts. Second, characterization of RS-exon reporters implicates exonic sequences as influencing choice of RS-exon usage. Using RS-exon swap and mutagenesis assays, we show exonic sequences can determine RS-exon inclusion. Finally, we provide evidence that splicing can suppress utilization of RP SDs to enable RS-exon expression. Overall, multiple factors can influence splicing of Drosophila RS-exons, which usually result in their complete suppression as zero-nucleotide RPs, but occasionally yield translated RS-exons.

Nuclear total RNA sequencing reveals primary sequence context of recursive splicing in long genes

BackgroundRecursive splicing (RS) is a mechanism to excise long introns from messenger RNA precursors. We focused on nuclear RNA, which is enriched for RS splicing intermediates and nascent transcripts, to investigate RS in the mouse brain.ResultsWe identified novel RS sites and discovered that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. We found that the primary sequence context, including the U1 snRNA binding site, the polypyrimidine tract, and a strong 3’ splice site, distinguishes the RS AGGT site from hundreds of non-RS AGGT sites in the same intron. Moreover, we uncovered a new type of exon-like RS events termed exonicRS.ConclusionsWe demonstrate that nuclear total RNA sequencing is an efficient approach to identify RS events. We find the importance of the primary sequence context in the definition of RS AGGT sites. The exonicRS may represent an intermediate stage of RS sites evolving into annotated exons. Overall, our findings provide novel insights into the mechanisms of RS in long genes.

Numerous recursive sites contribute to accuracy of splicing of long introns in flies

Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates which are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.