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PLoS Genet. 2018 Aug 27;14(8):e1007588. doi: 10.1371/journal.pgen.1007588. eCollection 2018 Aug.

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

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Department of Biology, Massachusetts Institute of Technology, Cambridge, United States of America.
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States of America.
Department of Computer Science, Stanford University, Stanford, United States of America.
Epigenetics and Stem Cell Biology Laboratory, Harvard Medical School, Boston, United States of America.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States of America.


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 that 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. A subset of recursive sites were validated by RT-PCR and sequencing. 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.

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