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.
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Extended data figures and tables
a, Identification of recursive splice sites by parsing alignments. RNA-seq reads were mapped to the genome using TopHat in a manner that allowed for novel splice junctions to be predicted. The alignments were then parsed for splice junction reads where the 5′ splice site mapped to an annotated 5′ splice site, but the 3′ splice site was unannotated. b, De novo identification of recursive splice sites. A database was generated in which each annotated 5′ splice site was spliced to all AG/GT sequences in an intron that did not correspond to an annotated 3′ splice site. All RNA-seq reads were aligned to this database and the alignments parsed to find cases where reads mapped perfectly with at least three distinct offsets and at least an 8 nt overhang.
a, Distribution of the number of ratchet points per recursive intron. b, Size distribution (log10(bp)) of all (red) and recursive (blue) introns. c, Size distribution (in kb) of the individual intron segments removed by recursive splicing binned by the number of segments per intron.
a–m, RT–PCR validation of ratchet points (red dots) from the indicated genes using primers in the upstream constitutive exon and flanking the putative ratchet points. The RP primers are expected to yield RT–PCR products if the constitutive exon is spliced to the ratchet point. The URP primers, which are upstream of each ratchet point, serve as negative controls. The identity of all RT–PCR products was verified by Sanger sequencing. Although the URP control RT–PCR reactions yielded a product for hppy RP1 and pum RP2, we were not able to generate sequence from them and therefore consider them to be amplification artefacts.
a, Examples of chromatin marks at the luna gene locus, which contains 5 recursive splice sites (red triangles) within a single long intron. b, Heat maps show relative ChIP-seq enrichment for H3K4me3 (top, red), H3K79me2 (middle, green) and H3K36me3 (bottom, blue), within 2 kb of the indicated gene features from 171 genes containing at least one ratchet point. Heat maps are centred around gene features, which include the transcription start site of the first exon (first exon, arrow), the 5′ splice site of the exon upstream of the recursive splice site (upstream exon, black rectangle), the ratchet point (red triangle), the 3′ splice site of the exon downstream of the recursive splice site (downstream exon, black rectangle), and the poly(A) site of the last exon (last exon, red octagon); the average exon of each gene feature is drawn to scale. Genes are sorted from top to bottom by decreasing expression level. For genes containing more than one ratchet point, the first, upstream, downstream and last exons are represented multiple times. c, Histogram illustrating the intron positions the ratchet points reside in based on RefSeq annotations.
This file contains Supplementary Figure 1 and full legends for Supplementary Tables 1-7. (PDF 8163 kb)
This table contains a summary of D. melanogaster RNA Sequencing Samples Generated for these studies – see Supplementary Information file for full legend. (XLSX 33 kb)
This table contains summary information for 197 D. melanogaster ratchet points – see Supplementary Information file for full legend. (XLSX 63 kb)
This table contains a comparison of ratchet points identified in this study and in Burnette et al. – see Supplementary Information file for full legend. (XLSX 43 kb)
This table contains a summary of recursive intron lariats identified from total RNA-Seq data – see Supplementary Information file for full legend. (XLSX 14 kb)
This table contains details of ratchet points experimentally validated in other Drosophila species – see Supplementary Information file for full legend. (XLSX 62 kb)
This table contains summary information for 5 human ratchet points– see Supplementary Information file for full legend. (XLSX 39 kb)
This table contains GO analysis of recursively spliced Drosophila genes – see Supplementary Information file for full legend. (XLSX 73 kb)
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Duff, M., Olson, S., Wei, X. et al. Genome-wide identification of zero nucleotide recursive splicing in Drosophila. Nature 521, 376–379 (2015). https://doi.org/10.1038/nature14475
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