Background

Splicing enhancers

       Sequences have been identified in several mammalian genes that reside at variable distances from splice sites yet are required for splicing to occur, either in vivo or in vitro. Although such splicing enhancers have been identified in both exons and introns, exonic splicing enhancers are generally better characterized, and are probably more common. Such exonic splicing enhancers (ESEs) activate nearby splice sites (both 5' and 3' splice sites) and promote the inclusion (vs. skipping) of exons in which they reside. Initially, ESEs were recognized as purine rich motifs containing repeated GAR (GAA or GAG) trinucleotides. However, many other sequences have now been shown to have enhancer activity (see Tacke and Manley 1999 for review).

       Many exonic splicing enhancers are bound and activated by one or more of several related splicing factors known as SR proteins. SR proteins contain either one or two RNA-binding domains and "RS" domains that are characterized by numerous arginine-serine dipeptide repeats. SR proteins are not only essential for splicing, but also for each of the first three recognizable steps of spliceosome assembly. In vitro, any one of the several SR proteins can restore splicing to a splicing extract lacking SR proteins. Thus, the essential functions of individual SR proteins in splicing are at least partially redundant. However, there is considerable specificity to the activation of splicing by SR proteins through exonic splicing enhancers. Individual SR proteins differ with respect to the sequence-specificity of their RNA-binding domains, and with respect to their ability to recognize and activate different exonic splicing enhancer sequences (e.g. Liu et al 1998, Schaal and Maniatis 1999b).

       The relationship between sequence-specific binding by SR proteins and the activation of splicing by exonic splicing enhancers is complex and incompletely understood. Both restoration of splicing and activation of some, but not all, enhancer-dependent splicing events by an SR protein lacking the RS domain has been reported (Zhu and Krainer 2000). Conversely, recruitment of the RS domain of SR proteins to an RNA by means of an unrelated RNA-binding domain has been also reported to promote enhancer-dependent splicing (Gravely and Maniatis 1998). Of greatest significance to this proposal are the observations that SR proteins show tissue- specific patterns of expression, and that different SR proteins work through different sequences (Liu et al. 1998). These facts support a model in which both constitutive splicing and enhancer- dependent splicing are dependent upon SR proteins bound to RNA. Although only a dozen or so splicing events have been shown to be enhancer-dependent, the existence of exonic splicing enhancers (ESEs) within constitutively spliced exons (Schaal and Maniatis 1999a) suggests the possibility that ESEs are ubiquitous, redundant, and required for all splicing events. It is estimated that as many as 15-20% of random sequences 20 nt. long contain a splicing enhancer (Blencowe 2000). Thus, it appears likely that many sequences may act as splicing enhancers. What is clear is that the motifs recognized by SR proteins are short and degenerate. Examples of these motifs (from Tacke and Manley 1999) are SRSASGA (where R=A or G; S=C or G) for ASF/SF2 and UGCNGYY (where Y=C or U and N is any base).

Exonic splicing enhancers (ESEs) in plant pre-mRNA splicing.

       Early research on plant pre-mRNA splicing emphasized the role of AU-rich or U-rich sequences within introns (see Simpson and Filipowicz 1996; Brown and Simpson 1998; Schuler 1998). It is clear that U-rich sequence elements play important roles in intron definition, and that exon-skipping is a less common outcome of mutagenesis than is true in animals, where introns tend to be larger and exon definition is probably a more common mode of splice site selection (Berget 1995). On the other hand, a number of recent results have shown a role for exon sequences in the selection of plant splice sites (Egoavil et al. 1997; Simpson et al. 1998; Simpson et al. 1999; McCullough et al. 1996).

       There are compelling reasons to believe that ESEs play an important role in plant splicing. SR proteins, the mediators of ESE activity in vertebrates, are highly conserved in plants. This pattern of conservation includes reactivity with the monoclonal antibody mAb104 (Lopato et al 1996) and extends to function. A mixture of Arabidopsis SR proteins (Lopato et al. 1996), and atRSZp22 in particular (Lopato et al 1999a) can complement SR-deficient mammalian splicing extracts. Furthermore, plant SR proteins can influence splice site choice in mammalian nuclear extracts (Lazar et al 1995), and regulate alternative splicing in vivo (Lazar and Goodman 2000; Lopato et al. 1999). Our own phylogenetic analysis of SR protein genes in complete genomes (Saccharomyces, Schizosaccharomyces, Caenhorabditis, Drosophila, Arabidopsis and Homo; Kumar and Mount, in preparartion) shows that Arabidopsis has more SR protein genes than any other known organism by a factor of two (20 genes in Arabidopsis vs. 9 in humans). Some of these genes encode highly similar pairs. In at least one case, that of the two SF2/ASF homologues atSRp30 and atSRp34/SR1, the two genes are expressed in distinct patterns during development (Lopato et al. 1999b), suggesting functional differentiation.

       In a preliminary analysis, we searched our database of 5249 exons from 1131 confirmed genes in chromosome II of Arabidopsis (see above) to identify all over-represented short oligomers. The most common 9-mer in this set matches the consensus [AG]AAGAAGA[AG], which is a near-perfect match to the known ESEs in Drosophila. The bulk of these 9mers occur 40-80bp from either the 3 or the 5 end of the exon, a fact that is consistent with their function as ESEs.

       No splicing event in Arabidopsis has yet been shown to be enhancer-dependent. However, exon sequences have been shown to contribute to splice site selection in the manner expected of an ESE (Egoavil et al. 1997), and the absence of documented enhancer-dependence can be readily attributed to the fact that detailed mutational analysis is necessary to show enhancer- dependence. There is also ascertainment bias. Very often the effects of mutations in exons are attributed to missense or nonsense when they may also effect splicing. For example, Liu et al. (2001) have shown that a nonsense mutation causing the skipping of BRCA1 exon 18 affects splicing in vitro, and that a miss-sense mutation at the same position also causes exon skipping. These same authors present a statistical analysis of 50 mutations that cause exon skipping in vivo (based on a list compiled by Valentine, 1998) that supports the possibility that they are mutations in ESEs. It is quite possible that effects of many mutations in plant ESEs on splicing have been missed merely because RNA was never examined. Indeed, several recent reviews emphasize the possibility that (human) mutations in ESEs may be much more common than is recognized (Blencowe 2000, Mount 2000, Maquat 2001).

References