Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans Ali R. Awan, Amanda Manfredo, and Jeffrey A. Pleiss1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853 Edited by Michael Rosbash, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved June 26, 2013 (received for review October 24, 2012) Alternative splicing is a potent regulator of gene expression that early functional exon skipping have been previously suggested: vastly increases proteomic diversity in multicellular eukaryotes environmentally or developmentally regulated exon skipping in and is associated with organismal complexity. Although alterna- a unicellular eukaryote, and conservation of a particular exon- tive splicing is widespread in vertebrates, little is known about the skipping pattern across eukaryotic kingdoms (11, 14). Although evolutionary origins of this process, in part because of the absence examples of exon skipping in a few unicellular eukaryotes have – of phylogenetically conserved events that cross major eukaryotic been published (15 17), to our knowledge there are no published clades. Here we describe a lariat-sequencing approach, which examples of either evolutionarily conserved or environmentally offers high sensitivity for detecting splicing events, and its appli- regulated exon-skipping events in any unicellular eukaryote. fi Schizosaccharomyces pombe cation to the unicellular fungus, Schizosaccharomyces pombe,an The unicellular ssion yeast, ,shares many of the hallmarks of alternative splicing in mammalian sys- organism that shares many of the hallmarks of alternative splicing tems. Nearly 50% of S. pombe genes contain an intron, and almost in mammalian systems but for which no previous examples of half of those contain multiple introns (18, 19). Moreover, the splice exon-skipping had been demonstrated. Over 200 previously un- site sequences found within S. pombe introns do not conform to the fi annotated splicing events were identi ed, including examples of tight consensus sequences seen in some other unicellular fungi, but GENETICS regulated alternative splicing. Remarkably, an evolutionary analy- rather are marked by a degeneracy more similar to that seen in sis of four of the exons identified here as subject to skipping in human introns (20, 21). Importantly, a single bona fide member S. pombe reveals high sequence conservation and perfect length of the serine/arginine-rich (SR) family of splicing regulators and conservation with their homologs in scores of plants, animals, and several SR-like proteins implicated in the regulation of alternative fungi. Moreover, alternative splicing of two of these exons have splicing are encoded within the S. pombe genome (22, 23). Fur- been documented in multiple vertebrate organisms, making these thermore, cis-acting sequence elements important in splicing reg- the first demonstrations of identical alternative-splicing patterns ulation in multicellular eukaryotes have been shown to modulate in species that are separated by over 1 billion y of evolution. the splicing efficiency of individual S. pombe introns (24), and in- deed nonendogenous plant and mammalian intron sequences have pre-mRNA splicing | post-transcriptional gene regulation | phylogeny been shown to be properly excised in S. pombe (25, 26). Never- theless, although instances of intron-retention have been docu- mented (27, 28), no examples of exon skipping have been described he protein coding regions of eukaryotic genes are typically in S. pombe. Surprisingly, two recent RNA-seq studies (29, 30) Tinterrupted by noncoding introns that must be removed to failed to detect any instances of exon skipping in S. pombe;however, produce a translatable mRNA. The removal of introns, catalyzed it was unclear whether this reflected an absence of such splicing by the spliceosome, offers a powerful opportunity for an organism events in S. pombe or a limitation of RNA-seq for detecting them. to regulate gene expression. In mammals, where individual genes are often interrupted by multiple introns, it is now abundantly Results clear that the process of intron removal provides a critical regu- To facilitate a deeper analysis of global splicing patterns in latory control point for both qualitative and quantitative aspects S. pombe that might uncover exon-skipping events, we developed of gene expression (1). By changing the identity of the exons that an alternative approach designed to concentrate sequencing are included within the final mRNA, the process of alternative efforts on the products of a splicing reaction. This approach splicing plays a critical role in expanding the diversity of proteins seeks to enrich, purify, and sequence the excised circular lariat that can be synthesized within a cell (2). Moreover, alternative RNAs that are a product of every splicing reaction. To stabilize splicing can direct the production of isoforms of genes that are these circular RNAs, the gene encoding the debranching enzyme directly targeted to cellular decay pathways, providing a mecha- (Dbr1) responsible for linearizing introns (31) was genetically nism to quantitatively regulate gene expression (3, 4). deleted from an S. pombe strain. Because environmental stresses In mammalian organisms the predominant form of alternative can stimulate splicing regulation even in budding yeast (32), the splicing is exon skipping, wherein different combinations of exons Δdbr1 S. pombe strain was exposed to a variety of environmental are included in the final transcript. In contrast, exon skipping is stresses, including various nutrient deprivations, temperature far less prevalent in simpler eukaryotes (5, 6); however, recent variations, and chemical exposures (SI Materials and Methods studies suggest that splicing in the last eukaryotic common an- cestor was similar in many respects to splicing in vertebrates, in so much as it was intron-dense (7–9), had degenerate splice site Author contributions: A.R.A., A.M., and J.A.P. designed research; A.R.A. and A.M. per- sequences (10), and likely had many of the proteins involved in formed research; A.R.A. contributed new reagents/analytic tools; A.R.A., A.M., and J.A.P. alternative splicing (11, 12). Intron density correlates positively analyzed data; and A.R.A. and J.A.P. wrote the paper. with the prevalence of alternative splicing across the eukaryotic The authors declare no conflict of interest. kingdoms (13), and thus it has been posited that the intron-rich This article is a PNAS Direct Submission. eukaryotic ancestor had alternative splicing, and may have had Data deposition: The data reported in this paper have been deposited in the Gene Ex- exon skipping. However, it is less clear whether the inferred an- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48594). cestral exon skipping was functional or represents “noise” that 1To whom correspondence should be addressed. E-mail: [email protected]. was later harnessed in the evolutionary lineages that led to mul- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ticellularity (13, 14). Two “smoking guns” that would corroborate 1073/pnas.1218353110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218353110 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 and Dataset S1, Table S1). Total RNA from each individual Lariat Sequencing Identifies Over 200 Previously Uncharacterized sample was pooled together for analysis. Splicing Events. Although over 80% of the lariat-sequencing reads Circular RNAs are retarded in their mobility relative to linear mapped to previously annotated introns, nearly 15% of the reads RNA and can be detected in 2D polyacrylamide gels (33) as an mapped to regions currently annotated as untranslated or protein- arc above a diagonal of linear RNAs (Fig. 1A). Importantly, coding (Dataset S1, Table S2). To determine whether these peaks these arcs were only detected in RNA from strains lacking might represent novel, unannotated splicing events, probabilistic functional Dbr1, suggesting they are predominantly populated by modeling was used to score putative splicing motifs surrounding excised lariats (Fig. S1). To increase the chances of identifying each peak in these regions. A Markov model derived from known exon-skipping events, gel conditions were optimized to recover intron sequences (35) allowed the quality of putative introns to be circular RNAs in a size range likely to include lariats that con- scored (SI Materials and Methods), enabling identification of over tained skipped exons. According to the most recent S. pombe 200 previously uncharacterized introns (Dataset S1, Table S5), the genome annotation, the shortest lariat that could be formed by majority of which have splice site sequences that are indistin- skipping a single exon is 89 nucleotides; as such, gel conditions guishable from canonical S. pombe introns (Fig. S2). were determined to optimize recovery of lariats ∼85 nucleotides Nearly half of the unique introns identified here are located or greater (Materials and Methods). within the UTRs of protein-coding genes, two of which are shown The circular RNA arcs were excised from the 2D gels and in Fig. 2A. For both the cnl2 and caf5 genes, the peaks lie com- converted into cDNA by random priming without prior debranch- pletely within the 5′ UTR of the transcripts. For both of these ing. The resulting cDNAs were sequenced using Illumina’s3G