Exon Inclusion Is Dependent on Predictable Exonic Splicing Enhancers

Abstract

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.The splicing together of exons during the maturation of mRNA from a primary transcript represents a step that is fundamental in the transfer of information from DNA to protein for most genes in higher eukaryotes. This process is catalyzed by a supramolecular particle known as the spliceosome, which serves as the site for the two transesterification reactions that remove an intron and connect two adjacent exons. The spliceosome is composed of five small nuclear RNA molecules and perhaps hundreds of proteins (20,23,33,55). Despite this complexity, much has been learned about the identity of these components, their function, and the sequence of events that culminates in exon joining (2,5,16,21). Less understood are the earliest events that initiate splicing, in which the boundaries of the intron that must be removed are identified. The absolute accuracy of this molecular recognition is critical to gene function and must depend on signals comprised of sequence and/or structural elements in the RNA substrate.In higher eukaryotes, several classes of splicing sequence elements can be distinguished based on their function and location; the splice sites themselves constitute one such class (31, 39). The 5Ј splice site is comprised of a 9-base consensus sequence that includes an almost universally conserved GU surrounded by additional nucleotides that are less well conserved. Similarly, the 3Ј splice contains a highly conserved AG surrounded by a less conserved C and G and an upstream tract of about 10 bases that is rich in pyrimidines. Splice site sequences from tens of thousands of introns have been used to define these two consensus sequences and to construct position-specific scoring matrices to evalu...

Positive selection acting on splicing motifs reflects compensatory evolution

“…Our previous experimental tests indicated that most ESEs in human exons are required for efficient splicing; i.e., extensive redundancy was not present (Zhang et al 2005a). That result, coupled with evidence for positive selection reported here, prompted us to search for evidence of compensation for changes that discourage splicing by changes that encourage it.…”

“…In our previous work (Zhang and Chasin 2004;Zhang et al 2005a), we defined putative splicing motifs as 8-mers; here they were converted to 6-mers to enable their amalgamation with analogous motifs described by the Burge laboratory (Fairbrother et al 2002;Wang et al 2004). The latter were defined using different strategies, and the sets from the two laboratories were given approximately equal weight in the combined sets used here.…”

“…We combined predicted exonic splicing signals from our previous studies, putative ESEs (PESEs), and putative ESSs (PESSs) with RESCUE-ESE signals and FAS-hex3 ESSs, respectively, from Burge and colleagues (Fairbrother et al 2002;Wang et al 2004;Zhang and Chasin 2004;Zhang et al 2005a). We merged the sequences from both groups to make a more complete list of splicing signals.…”

Splicing mutations in glycogen-storage disease type II: evaluation of the full spectrum of mutations and their relation to patients’ phenotypes

“…cgi), 21,22 RESCUE-ESE (http://genes.mit.edu/burgelab/rescue-ese/) 23 and PESX (http://cubweb.biology.columbia.edu/pesx/). 24,25 …”

Identifying Splicing Regulatory Elements with de Bruijn Graphs

“…The minigene-based technology utilizes the natural transcriptional and splicing machinery but concentrates only on a genomic segment of interest (Warner and Chamberlain, 2006). Several studies have exploited minigene technology for identifying SREs (Barash et al, 2010b;Ke and Chasin, 2010;Zhang et al, 2005;Zhang and Chasin, 2004).…”