Recursive splicing-a mechanism of intron removal with an unexplored role in the largest genomes.

Intron splicing is a critical step that pre-mRNA transcripts undergo to become mature mRNAs. Although long thought to occur in a single step, introns are now also known to be removed by a multi-step process called recursive splicing. In recursive splicing, the spliceosome removes the intron one segment at a time with segments defined by discreet sequences called recursive splice sites. As each segment is removed, the remaining downstream intronic sequence is brought into contact with the upstream exon. Recursive splicing can be detected through RNA-seq analysis because it produces a "sawtooth" pattern of read depth across intron length with peaks corresponding to sites in the ephemeral partially spliced introns where the remaining downstream intron segments contact the upstream exon. Recursive splicing can also be detected by RNA lariat sequencing and real-time imaging of single-cell transcriptional and splicing dynamics. These methods have been applied to fruit flies, humans, and mice, revealing that recursive splicing 1) increases in prevalence with intron length, and 2) increases splicing fidelity, particularly in long introns. However, intron lengths in the typically sized genomes of these model organisms fail to represent the diversity that exists across the tree of life. Species with gigantic genomes like salamanders and lungfishes have introns that are ten- to 50-fold longer. Future studies targeting recursive splicing in gigantic genomes will provide a unique perspective on its functional significance and will also reveal whether this splicing mechanism plays a role in overcoming constraints placed on transcriptional capacity and efficiency by enormous introns.