Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans

Abstract

Alternative splicing is a potent regulator of gene expression that vastly increases proteomic diversity in multicellular eukaryotes and is associated with organismal complexity. Although alternative splicing is widespread in vertebrates, little is known about the evolutionary origins of this process, in part because of the absence of phylogenetically conserved events that cross major eukaryotic clades. Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing. Remarkably, an evolutionary analysis of four of the exons identified here as subject to skipping in S. pombe reveals high sequence conservation and perfect length conservation with their homologs in scores of plants, animals, and fungi. Moreover, alternative splicing of two of these exons have been documented in multiple vertebrate organisms, making these the first demonstrations of identical alternative-splicing patterns in species that are separated by over 1 billion y of evolution.pre-mRNA splicing | post-transcriptional gene regulation | phylogeny T he protein coding regions of eukaryotic genes are typically interrupted by noncoding introns that must be removed to produce a translatable mRNA. The removal of introns, catalyzed by the spliceosome, offers a powerful opportunity for an organism to regulate gene expression. In mammals, where individual genes are often interrupted by multiple introns, it is now abundantly clear that the process of intron removal provides a critical regulatory control point for both qualitative and quantitative aspects of gene expression (1). By changing the identity of the exons that are included within the final mRNA, the process of alternative splicing plays a critical role in expanding the diversity of proteins that can be synthesized within a cell (2). Moreover, alternative splicing can direct the production of isoforms of genes that are directly targeted to cellular decay pathways, providing a mechanism to quantitatively regulate gene expression (3, 4).In mammalian organisms the predominant form of alternative splicing is exon skipping, wherein different combinations of exons are included in the final transcript. In contrast, exon skipping is far less prevalent in simpler eukaryotes (5, 6); however, recent studies suggest that splicing in the last eukaryotic common ancestor was similar in many respects to splicing in vertebrates, in so much as it was intron-dense (7-9), had degenerate splice site sequences (10), and likely had many of the proteins involved in alternative splicing (11,12). Intron density correlates positively with the prevalence of alternative splicing across the eukaryotic kingdoms (13), and thus it has...

Genome-wide discovery of human splicing branchpoints

“…Previous attempts to experimentally determine branchpoints has relied on laborious, low-throughput RT-PCR or 2D-gel purification of lariats (Gao et al 2008;Awan et al 2013) or identified several hundred branchpoints from large-scale RNA-sequencing atlases (Taggart et al 2012;Bitton et al 2014). In contrast, RNA CaptureSeq and RNase R digestion significantly enrich for intron lariats, allowing genome-wide branchpoint detection in wild-type cells without blocking intron debranching (Bitton et al 2014).…”

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

“…First, the lariat intron will more effectively report on splicing events that result in NMD-sensitive mRNA isoforms (Awan et al 2013). Second, the lariat intron reports on splicing efficiently, because every read from a lariat intron reflects a splicing event.…”

“…In studies of splicing from the perspective of excised introns, introns have been enriched by stabilization in a strain in which the gene DBR1 was deleted (Spingola et al 1999;Juneau et al 2007;Zhang et al 2007;Awan et al 2013;Bitton et al 2014;Stepankiw et al 2015); DBR1 encodes a debranchase that linearizes a lariat through hydrolysis of the unique 2 ′ -5 ′ phosphodiester bond-a prerequisite for rapid, exonucleolytic degradation (Chapman and Boeke 1991). Early microarray-based studies inferred the location of introns as those genomic regions in which RNA levels increased in the dbr1Δ strain, relative to a wild-type strain (Juneau et al 2007;Zhang et al 2007).…”

Circular RNAs: Unexpected outputs of many protein-coding genes

“…The mature circular RNA consisting of mrps16 exon 2 then accumulates in cells, while the double lariat from the re-splicing event and the skipped linear mRNA are both rapidly degraded. 41,88 Interestingly, there are other examples of exon skipping events in S. pombe, 89 yet none of these genes appear to generate circular RNAs. 41 This argues that the production of an exoncontaining lariat is not sufficient for circular RNA biogenesis, and the mechanisms by which cells determine when to re-splice to yield a circular RNA remain unclear.…”