Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin

Abstract

Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes. While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe. Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S. pombe pre-messenger RNAs (pre-mRNAs). Second, as in extracts from mammalian cells, ESE function in S. pombe is compromised by mutations and increased distance from the 3 -splice site. Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast. Finally, we have identified five natural purine-rich elements from S. pombe that promote splicing of our reporter pre-mRNAs. Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution.[Keywords: ESE; U2AF; SR protein; Srp1p; Srp2p; ASF/SF2] Supplemental material is available at http://www.genesdev.org. Pre-messenger RNA (pre-mRNA) splicing in all eukaryotes requires conserved intronic sequence elements located at the 5Ј-and 3Ј-splice sites and branchpoint for both accurate recognition of exon/intron boundaries and catalysis of the two transesterification reactions. Nevertheless, it was appreciated early on that intronic signals alone do not suffice for efficient splicing of some vertebrate pre-mRNAs (Reed and Maniatis 1986;Nelson and Green 1988). The discovery that positively acting elements within exons designated exonic splicing enhancers (ESEs) promote splicing of upstream introns exposed additional layers of complexity in metazoan 3Ј-splice site selection (Black 2003). ESEs are found both in premRNAs that are constitutively spliced (Mayeda et al. 1999;Schaal and Maniatis 1999) and those that are subject to regulated splicing (e.g., Ryner and Baker 1991;Tian and Maniatis 1992). A large and growing body of evidence indicates that exonic splicing enhancers are widely distributed among metazoans from flies to hu-

The Prp4 Kinase: Its Substrates, Function and Regulation in Pre-mRNA Splicing

“…Noteworthy here, U2AF 59 is essential for growth [56][57][58]. Moreover, there is some evidence that the recruitment of U2AF 59 to auxiliary factor dependent introns might be mediated by the SR protein Srp2, which has been shown above to be an in vitro substrate of Prp4 kinase [26]. However, we have no data, whether U2AF 59 dependent introns are also Prp4 kinase dependent introns.…”

“…Varying the kinase activity with an ATP analog in a fission yeast strain expressing an (analog-sensitive) asPrp4 kinase indicates that recruitment of Srp2 to nascent intron containing transcripts depends on Prp4 kinase activity (D. Eckert and N. F. Käufer, unpublished results). It has been shown that Srp2 promotes the splicing of reporter genes by binding to purine-rich exonic splicing enhancer (ESE) sequences [26]. Based on these observations, we hypothesize that phosphorylation within the SR elements of Srp2 by Prp4 kinase in the nucleus may serve to target Srp2 to ESE containing pre-mRNAs, thereby modulating their splicing efficiency.…”

A U1-U2 snRNP Interaction Network during Intron Definition

“…2F, lanes 4, 6, and 8), whereas the RS domain of Rsd1 alone was sufficient to bind to GST-SpPrp5 (lane 2). Another S. pombe SR protein, SRp2, which is also involved in splicing (40), was tested as a control for RS domain specificity and showed no detectable binding to GSTSpPrp5 (lane 12). Thus, SpPrp5 and Rsd1 interact primarily through their RS/RS-like domains.…”

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

“…Importantly, a single bona fide member of the serine/arginine-rich (SR) family of splicing regulators and several SR-like proteins implicated in the regulation of alternative splicing are encoded within the S. pombe genome (22,23). Furthermore, cis-acting sequence elements important in splicing regulation in multicellular eukaryotes have been shown to modulate the splicing efficiency of individual S. pombe introns (24), and indeed nonendogenous plant and mammalian intron sequences have been shown to be properly excised in S. pombe (25,26). Nevertheless, although instances of intron-retention have been documented (27,28), no examples of exon skipping have been described in S. pombe.…”