Complex RNA Secondary Structures Mediate Mutually Exclusive Splicing of Coleoptera Dscam1

Abstract

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

Cis mutagenesis in vivo reveals extensive noncanonical functions of Dscam1 isoforms in neuronal wiring

“…Considering the presence of dual docking sites and bidirectional base pairings in the exon 9 cluster of distantly related lepidopteran, coleopteran, and hymenopteran Dscam1 (Fig. 1 A) ( 29 , 30 ), we speculate that an analogous docking site may be located in the intron upstream of exon 9.1 of Drosophila Dscam1 . However, sequence alignment revealed only relatively limited conservation across Drosophila species (Fig.…”

“…Base pairings between the docking site and the selector sequence for the exon 9 cluster of lepidopteran Dscam1 were experimentally verified ( 29 ). Base pairings between the docking site and the selector sequence in the hymenopteran Apis mellifera and the coleopteran Dendroctonus ponderosae were predicted by comparative genome comparison and structural modeling ( 29 , 30 ). These competing base pairings are conserved, but the docking site and selector sequence are clade specific.…”

Recent advances in RNA structurome

“…Recently, a set of hidden RNA secondary structures, termed balancer RNA pairings, was found to drive the stochastic choice of the exon 6 cluster in Dscam1 (Dong et al, 2022). Moreover, such competing RNA structural mechanisms were later found and experimentally validated in exon clusters 4 and 9 of Dscam and in other insect genes such as 14-3-3ξ pre-mRNA (Figure 7C) (Dong et al, 2021;Hong et al, 2021;Yang et al, 2011;Yue et al, 2016). In addition to canonical splicing, a kind of non-canonical splicing called back splicing has been shown to be the fundamental mechanism for generating circular RNAs (Kristensen et al, 2019).…”

Cis mutagenesis in vivo reveals extensive noncanonical functions of Dscam1 isoforms in neuronal wiring

“…We have previously identified the docking site in the intron upstream of exon 10 of Drosophila Dscam1, which may pair with the selector sequence downstream of only a few exon 9 variants. (Hong et al, 2021;Yang et al, 2011) Considering the presence of dual docking sites and bidirectional base pairings in the exon 9 cluster of distantly related lepidopteran, coleopteran and hymenopteran Dscam1 (Figure S1A), (Dong et al, 2021;Yue et al, 2016) we speculated that an analogous docking site may be located in the intron upstream exon 9.1 of Drosophila Dscam1. However, sequence alignment revealed only relatively limited conservation across Drosophila (Figure S1B).…”