Splicing of designer exons reveals unexpected complexity in pre-mRNA splicing

Abstract

Pre-messengerRNA (mRNA) splicing requires the accurate recognition of splice sites by the cellular RNA processing machinery. In addition to sequences that comprise the branchpoint and the 39 and 59 splice sites, the cellular splicing machinery relies on additional information in the form of exonic and intronic splicing enhancer and silencer sequences. The high abundance of these motifs makes it difficult to investigate their effects using standard genetic perturbations, since their disruption often leads to the formation of yet new elements. To lessen this problem, we have designed synthetic exons comprised of multiple copies of a single prototypical exonic enhancer and a single prototypical exonic silencer sequence separated by neutral spacer sequences. The spacer sequences buffer the exon against the formation of new elements as the number and order of the original elements are varied. Over 100 such designer exons were constructed by random ligation of enhancer, silencer, and neutral elements. Each exon was positioned as the central exon in a 3-exon minigene and tested for exon inclusion after transient transfection. The level of inclusion of the test exons was seen to be dependent on the provision of enhancers and could be decreased by the provision of silencers. In general, there was a good quantitative correlation between the proportion of enhancers and splicing. However, widely varying inclusion levels could be produced by different permutations of the enhancer and silencer elements, indicating that even in this simplified system splicing decisions rest on complex interplays of yet to be determined parameters.

Identification and experimental validation of splicing regulatory elements in Drosophila melanogaster reveals functionally conserved splicing enhancers in metazoans

“…A previous study of SREs cautions that many computational predictions have been ''too successful,'' because now at least 75% of a typical human exon sequence can be shown to influence splicing (Zhang et al 2009). Our study indicates which of these many SREs are particularly relevant by identifying SREs that overlap between Drosophila and vertebrates.…”

“…Since splicing patterns can differ between tissue type and developmental stages, it is also necessary to study splicing regulation in diverse cellular contexts (Matlin et al 2005;Zhang et al 2009), taking into account the SREs' role in the pantheon of splice affecters.…”

“…The difference in the magnitude of enhancement between the tested ESEs and ISEs may be due to effects from local sequence context or relative position ( Fig. 4D; Goren et al 2006;Zhang et al 2009). …”

“…The specific combination of SREs and their distances from splice junctions contributes 4 These two authors contributed equally to this work. 5 to splicing outcome (Zhang et al 2009). In addition, current models for splice-site selection suggest that splice sites are recognized through interactions of the spliceosome across exons, termed ''exon definition,'' when exons are flanked by long introns and across introns, termed ''intron definition,'' when introns are short (Robberson et al 1990;Romfo et al 2000;Lim and Burge 2001;.…”

Splicing of designer exons informs a biophysical model for exon definition

“…More importantly this sequence has the property of not "creating" either a predicted ESE or a predicted ESS when all overlapping eight-mers formed by self-concatenation are considered (Zhang et al 2009). However, in the analysis presented here it appears to have weak silencing activity (see below); we therefore refer to it as the Reference Sequence (R), since the effects of the ESEs and ESSs used were evaluated by substituting them for the Reference Sequence.…”