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

Abstract

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 which 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. 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.Columns 8-10 -ie_count_ [timepoint]: count of the intron-exon junction reads for each of the labeling periods (summed across three replicates per labeling period) -for recursive sites, this overlaps the recursive site, while for the final segment this overlaps the 3' splice site.Columns 11-13 -ee_count_[timepoint]: count of the exon-exon junction reads for each of the labeling periods (summed across three replicates per labeling periods) -this includes junctions deriving from recursive intermediates, as outlined in the Methods.Column 14 -halflife: Half-life of the recursive segment computed using the junction dynamics approach described in Pai et al. 2017 (min).Column 15 -txn_to_three: Time to transcribe the remainder of the intron from the recursive site to the 3' splice site (min).

The Chimonanthus salicifolius genome provides insight into magnoliid evolution and flavonoid biosynthesis

“…Mutations that occurred in the recursive splicing sites resulted in many human diseases (Chabot and Shkreta, 2016). The recursive splicing is a splicing phenomenon difficult to capture, and requires nascent RNA sequencing, which can profile pre-mRNA transcripts shortly after they are transcribed (Pai et al, 2018). With the data for designed transcriptomic experiments, more characteristics for the long genes (such as recursive splicing and other mechanisms) in the genomes of magnoliids could be explored in future.…”

Recursive splicing is a rare event in the mouse brain

“…Given the high conservation of RS mechanism among species (Duff et al, 2015;Joseph et al, 2018;Pai et al, 2018;Sibley et al, 2015;Zhang et al, 2018), we first investigated the sequence conservation of the 600 nt regions surrounding the RS sites. In agreement with previous studies(Duff et al, 2015; Joseph et al, 2018;Pai et al, 2018;Sibley et al, 2015), the AGGT motif at the RS sites is highly conserved across 60 vertebrate genomes, showing high phylogenetic pvalue scores (phyloP scores) compared to upstream and downstream regions ( Figure 4A). The RS introns, showing a median length of 267 kb and a mean length of 382 kb, were significantly longer than introns transcribed in the mouse cerebral cortex ( Figure 4B).…”

“…The RS is a splicing mechanism that is specific to long introns (Burnette et al, 2005;Cook-Andersen and Wilkinson, 2015;Duff et al, 2015;Hatton et al, 1998;Hayashi et al, 2018;Joseph et al, 2018;Kelly et al, 2015;Pai et al, 2018;Sibley et al, 2015;Zhang et al, 2018). This mechanism excises a long intron into several smaller segments as opposed to in a large single unit.…”

“…Whole-cell ribosomal RNA-depleted total RNA-seq and nascent RNA-seq have been used to capture RS splicing intermediates and nascent transcripts to identify RS events (Duff et al, 2015;Hayashi et al, 2018;Joseph et al, 2018;Pai et al, 2018;Sibley et al, 2015;Zhang et al, 2018). Nevertheless, the efficiency of capturing RS splicing intermediates and nascent transcripts using these methods is relatively low.…”

“…Meanwhile, little is known about why some long introns use the RS mechanism for splicing, while other long introns do not. Furthermore, RS utilizes the splicing mechanism of exon definition (Joseph et al, 2018;Pai et al, 2018;Sibley et al, 2015;Zhang et al, 2018), which requires an RS exon downstream of the AGGT site. Although RS exons are typically spliced from mature transcripts, multiple lines of evidence indicate that some RS exons can be retained in mature mRNAs (Joseph et al, 2018;Sibley et al, 2015).…”