Global analysis of trans-splicing in Drosophila

Abstract

Precursor mRNA (pre-mRNA) splicing can join exons contained on either a single pre-mRNA (cis) or on separate pre-mRNAs (trans). It is exceedingly rare to have trans-splicing between protein-coding exons and has been demonstrated for only two Drosophila genes: mod(mdg4) and lola. It has also been suggested that trans-splicing is a mechanism for the generation of chimeric RNA products containing sequence from multiple distant genomic sites. Because most high-throughput approaches cannot distinguish cis- and trans-splicing events, the extent to which trans-splicing occurs between protein-coding exons in any organism is unknown. Here, we used paired-end deep sequencing of mRNA to identify genes that undergo trans-splicing in Drosophila interspecies hybrids. We did not observe credible evidence for the existence of chimeric RNAs generated by trans-splicing of RNAs transcribed from distant genomic loci. Rather, our data suggest that experimental artifacts are the source of most, if not all, apparent chimeric RNA products. We did, however, identify 80 genes that appear to undergo trans-splicing between homologous alleles and can be classified into three categories based on their organization: (i) genes with multiple 3' terminal exons, (ii) genes with multiple first exons, and (iii) genes with very large introns, often containing other genes. Our results suggest that trans-splicing between homologous alleles occurs more commonly in Drosophila than previously believed and may facilitate expression of architecturally complex genes.

Fig. 1.

Deep sequencing to search for trans-spliced genes and chimeric RNAs. Sequencing libraries were prepared from poly(A)-selected RNA from F1 hybrids of D. melanogaster and D. sechellia, and from a mixture of parental RNA (control). Libraries were subjected to paired-end deep sequencing, and species-specific sequence reads were identified by comparing genomic alignments. Sequence mate-pairs in which both reads mapped to the same (cis) or different (trans) species were mapped to genes to identify pairs indicative of splicing.

Fig. 2.

Trans-splicing of mod(mdg4) and lola. The sequencing results obtained for mod(mdg4) (A) and lola (B) are shown. The horizontal gray line separates the sense and antisense exons of mod(mdg4). The 3′ terminal exon groups for which deep sequencing data support trans-splicing (green), only cis-splicing (red), or are not expressed in the hybrid (gray) are shown. Isoforms for which trans-splicing was previously reported are depicted with an asterisk. The number of cis– (red) and trans– (green) mate-pairs observed for each isoform of mod(mdg4) and lola are shown (bar graphs).

Fig. 3.

Examples of newly identified trans-spliced genes. Trans-splicing was validated using RT-PCR with primers specific to D. melanogaster (red) and D. sechellia (blue). Trans-splicing is validated by the presence of RT-PCR products when using opposite species forward and reverse primers with hybrid, but not mixed control (Mix) cDNA. Several clones of these putative trans-splicing products were sequenced to verify a clean transition of species-specific sequences at splicing junctions. (A) CG42235 contains a set of common 5′ exons which are trans-spliced to multiple alternative 3′ terminal exon groups. (B) The ome gene has multiple alternative transcription initiation exons which are trans-spliced to a set of common 3′ terminal exons. (C) Nmdmc is an example of trans-splicing of nested genes, as the gene Rel is located within the intron.