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.

Keywords: phylogeny; post-transcriptional gene regulation; pre-mRNA splicing.

Fig. 1.

Purification and sequencing of excised lariat introns. (A) Stabilization of lariat RNAs is achieved by genetically deleting the gene encoding the debranching enzyme, Dbr1. Total cellular RNA isolated from a Δdbr1 strain grown under a variety of conditions was pooled and subjected to 2D gel electrophoresis, allowing for separation of linear RNAs from circular lariats. (B) Lariat RNAs recovered from the 2D gel were sequenced and aligned to the S. pombe genome. Read density plots are shown near the known introns in the sim4 and rpl4301 genes. Arrows indicate locations of primers used for confirmation. PCR products generated using either genomic DNA or cDNA as a template confirm the splicing of these introns. Locations of the unspliced and spliced products are noted.

Fig. 2.

Lariat sequencing identifies over 200 unannotated introns. (A) Read density plots of previously uncharacterized introns in the cnl2 and caf5 genes. Cartoons of the currently annotated coding regions are indicated below. The sequences corresponding to the putative branch point, 5′ and 3′ splice sites are indicated, as are the consensus sequences in known S. pombe introns (60). Flanking PCR demonstrates removal of the introns in cDNA from wild-type cells, but a block to splicing in cDNA from cells lacking the splicing factor, smd3 (Δsmd3 cDNA). (B) Read density plots of unannotated introns in the coding regions of the cys11 and rad8 genes. Intron length is indicated by arrows. Flanking PCR using cDNA from wild-type cells reveals an intermediate amount of splicing of these introns. In the background of a strain lacking the NMD factor upf1 (Δupf1 cDNA), the spliced isoform of rad8 is stabilized.

Fig. 3.

Examples of constitutive and inducible exon skipping. (A) Read density plots surrounding alternatively skipped exons (red) within the srrm1, alp41, and qcr10 transcripts. Peaks are colored red where the reads overlap with the skipped exon, and gray where they overlap with the flanking introns. PCR products generated using either genomic DNA or cDNA from wild-type cells grown under normal conditions are indicated; cDNA from wild-type cells exposed to either heat or cold shock are also indicated. Locations of the unspliced, spliced, and exon-skipped (*) products are noted (SI Materials and Methods and Fig. S11). (B) Flanking PCR using cDNA from a time-course of heat shock (alp41) or cold shock (qcr10) demonstrates the stress-induced alternative splicing of these transcripts.

Fig. 4.

Evolutionary conservation of skipped exons in the srrm1 and alp41 genes of S. pombe. (Left) Pruned phylogenetic trees depicting model species that have an ortholog of the skipped S. pombe exons in srrm1 (Upper) or alp41 (Lower), drawn according to NCBI taxonomy relationships (61). Full trees are shown in Fig. S10. Vertebrate branches are colored red and plant branches are colored green. (Right) For the srrm1 and alp41 genes, the two alternate S. pombe isoforms corresponding to the skipping events are shown with the corresponding isoforms from Ensembl (human) or UniProt (mouse) shown above. Only the first six exons of the human ortholog of the srrm1 gene are shown. The evolutionarily conserved exons are shown in red; intron lengths are shown in Fig. S10. Below the gene diagrams are shown the peptide translations of the S. pombe skipped exons and below that a peptide motif constructed from a multiple sequence alignment of the translated orthologous exons from all species for which an orthologous exon was found (60).