Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures

Abstract

Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons--the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA.

Figure 1. The D. melanogaster Dscam Gene and Insect Phylogeny

(A) Organization of the D. melanogaster Dscam gene. Dscam contains 115 exons, 95 of which are alternatively spliced. The exon 4, 6, and 9 clusters contain 12, 48, and 33 alternative exons, respectively, that each encode variable immunoglobulin domains. The exon 17 cluster contains two exons that encode alternate versions of the transmembrane domain. The exons within each cluster are alternatively spliced in a mutually exclusive manner. The exon 6 cluster is enlarged to depict its organization. (B) Dendrogram of the phylogenetic relationship among the insects used in this study. The tree represents the estimated evolutionary distance of each organism in millions of years. These organisms represent four major orders of Insecta—Diptera, Lepidoptera, Hymenoptera, and Coleoptera—which are each shaded in different colors.

Figure 2. The Docking Site

The nucleotide sequence alignment of the docking sites of 15 insects. The most common nucleotide at each position is shaded. The docking site consensus is represented as a pictogram (bottom). The height of each letter represents the frequency of each nucleotide at that position.

Figure 3. Conservation of Selector Sequences

Alignment of eight of the selector sequences and their locations with the exon 6 cluster are depicted. The most common nucleotides at each position are shaded.

Figure 4. The D. melanogaster Selector Sequence Consensus

(A) The 48 selector sequences and flanking sequence were aligned together. The most frequent nucleotides in the central portion of the alignment are highlighted. (B) The alignment was used to generate a selector sequence consensus.

Figure 5. The Docking Site and Selector Sequences Consensus Are Complementary

The docking site consensus sequence is complementary to the central 28 nucleotides of the selector sequence consensus. The most frequent nucleotide at each position of the selector sequence is complementary to the docking site.

Figure 6. Conservation of the Docking Site:Selector Sequence Secondary Structures

(A) The RNA secondary structures of the proposed interactions between the docking site and the exon 6.5 and 6.12 selector sequences are shown for each Drosophila species. Although the precise structure is not absolutely conserved in each species, similar structures have the potential to form. (B) The RNA secondary structures of the docking site with the exon 6.4, 6.5, 6.9, and 6.10 selector sequences from A. meliffera are shown. These examples demonstrate that the two nucleotides in the docking site that are invariant in all other species (shaded in green) engage in base-pairing interactions with the selector sequences. These compensatory mutations provide additional evidence supporting the proposed docking site:selector sequence interactions.

Figure 7. Model for the Mechanism of Dscam Exon 6 Mutually Exclusive Splicing

A model of the Dscam exon 6 cluster is depicted in which only variable exons 6.36, 6.37, and 6.38 are shown. A key component of this model is that a splicing repressor functions to prevent the exon 6 variants from being spliced together (green oval). In order for an exon 6 variant to be included in the Dscam mRNA, the selector sequence upstream of the exon must interact with the docking site. For example, if exon 6.36 is to be included (left), the selector sequence upstream of exon 6.36 will interact with the docking site. Likewise, if exon 6.37 is to be included, the selector sequence upstream of exon 6.37 will interact with the docking site. By some unknown mechanism, the docking site:selector sequence interaction inactivates the splicing repressor on the downstream exon and, consequently, activates the splicing of the downstream exon 6 variant to exon 5. Subsequently, the exon that is joined to exon 5 can only be spliced to constitutive exon 7 because the remaining exon 6 variants are actively repressed by the splicing repressor. As a result, only one exon 6 variant is included in the mRNA.