Recursive splicing is a process in which large introns are removed in multiple steps by re-splicing at ratchet points—5′ splice sites recreated after splicing1. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene1 and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing2,3. Here we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila. Finally, we identify four recursively spliced human genes, one of which is also recursively spliced in Drosophila. Together, these results indicate that recursive splicing is commonly used in Drosophila, occurs in humans, and provides insight into the mechanisms by which some large introns are removed.
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This work was supported by NHGRI grant U54HG006994 to S.E.C. (PI) and B.R.G. (co-PI), and R01GM095296 to B.R.G.
Extended data figures
Extended data tables
This table contains a summary of D. melanogaster RNA Sequencing Samples Generated for these studies – see Supplementary Information file for full legend.
This table contains summary information for 197 D. melanogaster ratchet points – see Supplementary Information file for full legend.
This table contains a comparison of ratchet points identified in this study and in Burnette et al. – see Supplementary Information file for full legend.
This table contains a summary of recursive intron lariats identified from total RNA-Seq data – see Supplementary Information file for full legend.
This table contains details of ratchet points experimentally validated in other Drosophila species – see Supplementary Information file for full legend.
This table contains summary information for 5 human ratchet points– see Supplementary Information file for full legend.
This table contains GO analysis of recursively spliced Drosophila genes – see Supplementary Information file for full legend.