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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...

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The trp RNA-binding attenuation protein (TRAP) functions in many Bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary structural elements in the 5′ leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies, corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced fit conformational change are discussed. Keywordscalorimetry; binding-coupled protein folding; allosteric regulation; oligomer; trp RNA-binding attenuation protein; induced fit; pre-existing conformational equilibriumThe undecameric (11-mer) trp RNA-binding attenuation protein (TRAP) is responsible for controlling the transcription (1-5), and in some cases the translation (6-11), of the genes responsible for tryptophan biosynthesis in many Bacilli. Transcriptional regulation of the trp operon in these Bacilli is achieved through attenuation, in which competing secondarystructural elements in the 5′ leader region of the nascent mRNA control the extent of transcriptional read-through of the structural genes (3-5). TRAP exercises transcriptional control by influencing the formation of these secondary-structural elements through tryptophan-dependent binding to the RNA. When tryptophan is limiting, TRAP is inactive and * Corresponding author contact information: Phone: 614-292-1377, FAX: 614-292-6773, Email: foster.281@osu does not bind to the RNA. This allows a stable anti-terminator hairpin to form, promoting transcriptional read-through of the entire operon. However, when the intracellular tryptophan level is sufficiently high, TRAP binds to tryptophan and becomes activated to bind to eleven triplet repeats of (G/U)AG's present in the 5′ leader region of the mRNA. When TRAP is bound, the anti-terminator RNA structure cannot form, allowing preferential formation of the term...