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Short cryptic exons mediate recursive splicing in Drosophila


Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect ‘zero-nucleotide exons’, whose sequential processing subdivides removal of long host introns. We used CRISPR–Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40–80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.

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Fig. 1: RP-donor mutants of Ubx and kuz are strong loss-of-function alleles.
Fig. 2: Molecular evaluation of RP mutants reveals existence of cryptic exons.
Fig. 3: Genome-wide annotation of novel intronic RPs and RP exons.
Fig. 4: Genome-wide identification of RP-associated cryptic exons.


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We thank the Bloomington Drosophila Stock Center and the Developmental Studies Hybridoma Bank for fly stocks and antibodies used in this study. S.K. was supported by the Mochida Memorial Foundation for Medical and Pharmaceutical Research. Work in E.C.L.’s group was supported by the National Institutes of Health (R01-NS083833 and R01-GM083300) and MSK Core Grant P30-CA008748.

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Authors and Affiliations



B.J. generated the Ubx-RP allele, performed all of the immunostaining, molecular analysis and computational studies. S.K. generated the Bx-RP and kuz-RP alleles. E.C.L. conceived the project, coordinated the studies, and wrote the paper with input from the coauthors.

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Correspondence to Eric C. Lai.

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Integrated supplementary information

Supplementary Figure 1 Evidence and mechanistic models for recursive splicing.

(a) Sawtooth RNA-seq patterns are indicative of recursive splicing intermediates. Generally, RNA-seq coverage in introns is reflective of nascent transcription and resembles right-angled triangles, with highest coverage at the 5’ end and lowest at the 3’ end of the intron. However, introns that undergo recursive splicing consist of multiple intronic segments, each with its own right-angled triangle coverage, producing a sawtooth pattern. This property has been exploited to infer recursive splicing and annotate RPs. (b) Models for processing introns with RPs. It is conceivable that introns that contain RPs will be processed in one of two ways. First, the RP is utilized constitutively (path 1) and the intron is removed in two sequential steps. Second, the RP is skipped such that the entire intron is spliced out in one step (path 2). (c) A molecular model for recursive splicing. We propose that recursive splicing proceeds by first defining a cryptic RP exon, which is specified by the RP splice acceptor and a downstream cryptic splice donor. Definition of the cryptic RP exon allows removal of the first intron segment and production of the recursive intermediate. In the second splicing reaction, we propose that the regenerated RP splice donor outcompetes the cryptic splice donor, thereby removing the whole intron and ligating neighboring exons.

Supplementary Figure 2 Genes with RPs were manipulated to identify cryptic exons.

(a) UCSC genome browser screenshots display the three genes (Bx, kuz, and Ubx) manipulated in this study, including the approximate genomic locations of RPs within long host introns. (b) Nature of mutant alleles along with sequence alignments. (c) UCSC genome browser nucleotide-level screenshots of mutated RPs (grey highlight) along with cryptic exons detected in mutants (yellow highlight). *Ubx[RP] is an insertion mutant, which separates the RP splice acceptor and donor sites by 38nt.This 38nt insertion is retained in mutant animals. However, bioinformatic analysis has identified a naturally occurring cryptic exon and splice donor as indicated.

Supplementary Figure 3 kuz cryptic exon is retained in S2-R+ cells.

Sashimi plots were used to display the usage of the cryptic exon in modENCODE total RNA-seq data from S2-R+ cells, L1 stage larvae and 22-24hr embryos. Spliced reads can only be detected into and out of the cryptic exons in S2-R+ cells, suggesting selective inclusion and/or stabilization here.

Supplementary Figure 4 Quantification of relative cryptic exon inclusion ratios from wild-type and mutant recursive splicing minigenes.

x and kuz minigenes that contained the indicated mutations (see Fig 2E, G) were transfected into S2 cells and subjected to rt-PCR analysis. Relative exon inclusion was calculated by normalizing the intensity of the mRNA band to all indicated bands in the same lane, and then scaled to total expression observed in wt lane. Representative gels are shown (see also main Figure 2).

Supplementary Figure 5 Nascent RNA-seq datasets are more suited for detection of RPs.

(a, b) Nascent RNA-, total RNA- and mRNA-seq datasets from S2 cells were evaluated for different criteria. (a) Nascent RNA datasets have higher coverage at intronic loci. Long intronic segments - with no overlapping genes - were identified and reads mapping to these regions were summed and normalized. (b) Junction spanning reads that mapped to RPs identified in Duff et al. were summed and normalized. (c) Junction spanning reads found from all nascent RNA-seq and GRO-seq were merged and those with 3’ ends mapping to intronic regions were stratified by junction split (intron length) into three categories. For each category, pie charts were drawn to indicate tetranucleotide distributions at the 3’ junction end. Note that AGGT, which resembles minimal ratchet point sequences are enriched only within the long intron category.

Supplementary Figure 6 Novel RPs share sequence, structural, and evolutionary properties of known RPs.

(a) Comparison of average phyloP scores for “0-nt” RPs. New RPs were grouped into two categories based on whether they had sawtooth patterns in RNA-seq data or not. (b) Average length of host introns for RPs found in Duff et all and this study. (c) Sequence logos for categorized RPs.

Supplementary Figure 7 Example of intronic RP and RP-exon annotation in the msi gene.

The BLAT tool in UCSC genome browser was used to map RNA-seq reads to musashi (msi). 5’ ends of reads map to a msi 5' exon (zoomed in shot highlighted in red). 3’ ends of one read maps to an intronic RP (blue highlight) and a zoomed in nucleotide-level screenshot is included in blue. 3’ ends of the other read maps to an RP exon (green highlight) and a zoomed in nucleotide-level screenshot is included in green. Note the RNA-seq coverage in screenshots and that RP exons have distinct exon coverage, whereas intronic RPs have sawtooth coverage pattern. The core AGGT splice acceptor-donor pairs are marked in gray, while the larger splice consensus motifs are highlighted in yellow.

Supplementary Figure 8 Conservation and coding properties of RP exons by subcategory.

(a) Distribution of RP exons according to location in gene models. (b) RP exons were divided according to their location in 5’UTR, CDS, and 5’UTR/CDS (ones that contained alternate 5’UTR/start sites). The fully coding RP exons have a high level of evolutionary conservation, and the set with partial coding potential exhibit an intermediate level of conservation.

Supplementary Figure 9 Positional bias and conservation of cryptic donors stratified by splice scores.

(a) Splice donors found downstream of RPs (cryptic splice donors) or 1000 control AGGTs sites were grouped based on NNSPLICE splice site strength. Plotted is the positional bias of splice donor site position relative to ratchet points. Substantial enrichment is observed in the ~40-80 window downstream of ratchet points, but not control AGGT sequences, at NNSPLICE scores down to 0.5-0.6 (b) Average phyloP scores of splice donor sites downstream of ratchet points (RPs) segregated into those that are <100nt from RPs and >100nt away from RPs. Clear local conservation is observed amongst groups of cryptic donors scored down to ~0.6. (c) Table showing the number of non-overlapping RPs with cryptic splice sites grouped by splice site score.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Note 1

Reporting Summary

Supplementary Table 1

Supplementary Dataset 1

Uncropped gels reported in this study.

Supplementary Dataset 2

Summary of datasets analyzed in this study.

Supplementary Dataset 3

Annotations of Drosophila intronic RPs and RP exons.

Supplementary Dataset 4

Examples of Drosophila RP exons. Shown are UCSC Genome Browser screenshots with gene models and RNA-seq evidence at cassette exons that can regenerate conserved 5’ splice sites.

Supplementary Dataset 5

UCSC genome browser screenshots of intronic RPs and their associated cryptic exons. Shown are 50 examples of intronic RPs with high-scoring cryptic splice donors; the inferred cryptic exons are highlighted in red.

Supplementary Dataset 6

Gene Ontology analyses of Drosophila genes undergoing recursive splicing. Hypergeometric tests and Bonferroni corrections were used to obtain adjusted P values.

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Joseph, B., Kondo, S. & Lai, E.C. Short cryptic exons mediate recursive splicing in Drosophila . Nat Struct Mol Biol 25, 365–371 (2018).

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