Recursive splicing (RS) is a mechanism to excise long introns from messenger RNA precursors. New York Institute of Technology researchers focused on nuclear RNA, which is enriched for RS splicing intermediates and nascent transcripts, to investigate RS in the mouse brain. The researchers identified new RS sites and discovered that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. Moreover, they found that the primary sequence context, including the U1 snRNA binding site, the polypyrimidine tract, and a strong 3′ splice site, determines RS sites in long introns. The researchers also uncovered a new type of exon-like RS events termed exonicRS that may represent an intermediate stage of RS sites evolving into annotated exons. Furthermore, the strengths of 5′ splice sites are able to classify RS events into RS sites and exonicRS. Overall, these findings provide mechanistic insights into the splicing and evolution of long genes.
Nuclear total RNA-seq is efficient to identify recursive splicing (RS) events
(a) Schematic of the two features of RS – the saw-tooth pattern (red triangles) and the RS junction read. (b) Schematic of the isolation of different types of RNA. (c) Sequencing profile at Hs6st3 locus. r1, replicate 1. kb, kilobases. (d) Boxplot of normalized numbers of RS junction reads at Hs6st3 RS site. RPM, reads per million uniquely mapped reads. *, P = 0.03, one-tailed t-test. (e) Schematic of the pipeline utilizing nuclear total RNA seq data to identify RS sites. (f) Sequencing profile at Lsamp locus. Green arrows indicate the four novel RS sites. (g) Bar plot of RS sites identified utilizing the two sequencing methods in the mouse cortex.