A Subset of SR Proteins Activates Splicing of the Cardiac Troponin T Alternative Exon by Direct Interactions with an Exonic Enhancer

Abstract

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.Pre-mRNA splicing involves two transesterification reactions mediated by the spliceosome (11, 21). The mechanisms by which exon-intron borders are first defined within complex pre-mRNAs remain largely unknown. Although splice donor and acceptor sequences are fairly well conserved among premRNAs, these elements are not sufficient to distinguish bona fide splice sites from unused cryptic splice sites present in many metazoan pre-mRNAs. Because splicing is so precise, additional controls for splice site selection must be present elsewhere in the pre-mRNA.Results with vertebrate and invertebrate experimental systems have demonstrated that sequences within exons, exclusive of splice junctions, can play a role in splice site selection (13). One positively acting splicing element is a 13-nucleotide repeat in the female-specific exon of the Drosophila doublesex (dsx) gene. Two proteins, transformer-2 and transformer, promote utilization of an upstream 3Ј splice site via direct interactions with the repeats (12,15,22). Purine-rich splicing elements, called splicing enhancers, have recently been identified in several vertebrate exons (1,6,16,19,28,31,33,35,36) and in the dsx female-specific exon (20). Splicing enhancers are required for efficient splicing of the resident exon, and several have been shown to enhance splicing of heterologous exons (16,19,33,35,36). With one exception (16), splicing enhancers appear to activate splicing of the upstream intron (35). The enhancer in the fibronectin ED1 exon has been shown to promote binding of the U2 small nuclear ribonucleoprotein particle (snRNP) to the upstream branch site (19), which is consistent with this observ...

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

“…3A,B) dramatically reduce Srp2p binding (data not shown). Taken together, these results provide strong evidence that the ability of these purine-rich elements to promote splicing in S. pombe is due to their ability to bind Srp2p, as observed for a subset of mammalian SR protein family members (Ramchatesingh et al 1995).…”

Combinatorial control of a neuron-specific exon

“…In addition to the 39 splice site and the multiple elements of the downstream enhancer, there is another element affecting splicing of the N1 exon+ This sequence is at the 59 end of the N1 exon itself and includes the element GAGGAAGG+ Similar purine-rich elements act as exonic splicing enhancers in other exons (Lavigueur et al+, 1993;Sun et al+, 1993;Tian & Maniatis, 1993;Staknis & Reed, 1994;Ramchatesingh et al+, 1995;Yeakley et al+, 1996)+ When placed by itself in the test exon, this sequence had only a small effect on splicing+ However, activation of the test exon by an intronic enhancer always increased in the presence of this exonic element (Fig+ 7)+ This was most striking for clone 4-187, containing the triple GGGGGCUG repeat, in 3T3 and HEK293 cells (Fig+ 8)+ With this combination of exonic and intronic elements, splicing increased fourto fivefold over either element alone+ The identification of this N1 exonic element, which was missed in earlier mutagenesis studies of the c-src gene, underscores The combination of exonic and intronic enhancer elements has also been described in the regulation of exon 5 of the cardiac troponin T transcript (Ryan & Cooper, 1996)+ In this case again, the exonic element is a general splicing enhancer, whereas the intronic elements determine the tissue specificity+ The intronic GGGGGCUG element in the src transcript is thought to bind to hnRNPs H and F in vitro (Min et al+, 1997;Chou et al+, 1999), whereas the exonic element is similar to sequences known to bind to the proteins SF2/ASF and Tra-2 (Sun et al+, 1993;Tacke & Manley, 1995;Liu et al+, 1998;Tacke et al+, 1998)+ It is not clear whether the proteins bound to these two RNA elements directly contact each other or instead independently interact with components of the spliceosome to alter their assembly+ It will be interesting to examine the proteins binding to these src regulatory elements to determine their interactions with each other and to look at their effect on the assembly of other splicing factors+…”

A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.

“…Exonic splicing enhancers are sequences, found in certain exons, that can stimulate spliceosome assembly at the upstream 3' splice site of the exon (Fu et al 1991;Lavigueur et al 1993;Sun et al 1993;Watakabe et al 1993;Caputi et al 1994;Dirksen et al 1994;Staknis and Reed 1994;Tanaka et al 1994;Tian and Maniatis 1994;Ramchatesingh et al 1995;Wang et al 1995). Exonic enhancers are usually purine-rich sequences whose ac-…”