The evolutionary landscape of intergenic trans-splicing events in insects

Abstract

To explore the landscape of intergenic trans-splicing events and characterize their functions and evolutionary dynamics, we conduct a mega-data study of a phylogeny containing eight species across five orders of class Insecta, a model system spanning 400 million years of evolution. A total of 1,627 trans-splicing events involving 2,199 genes are identified, accounting for 1.58% of the total genes. Homology analysis reveals that mod(mdg4)-like trans-splicing is the only conserved event that is consistently observed in multiple species across two orders, which represents a unique case of functional diversification involving trans-splicing. Thus, evolutionarily its potential for generating proteins with novel function is not broadly utilized by insects. Furthermore, 146 non-mod trans-spliced transcripts are found to resemble canonical genes from different species. Trans-splicing preserving the function of 'breakup' genes may serve as a general mechanism for relaxing the constraints on gene structure, with profound implications for the evolution of genes and genomes.

Figure 1. Identification of intergenic trans-splicing events in eight species across five orders of class Insecta.

The branching order and divergence times are derived from the TimeTree database. TS, intergenic trans-splicing events.

Figure 2. Characterization of intergenic trans-splicing events in insects.

(a) Schematic diagram of four organizational types of donor and acceptor genes involved in intergenic trans-splicing. The arrows denote the direction of transcripts and their relative positioning on the chromosome; donor genes are in red and acceptor genes are in blue. The counts for each type are indicated in parentheses to the right. DfChr, different chromosomes/scaffolds; DfStr, different strand; SmAD, same chromosome with acceptor upstream of donor; SmDA, same chromosome with donor upstream to acceptor. (b) Distances between canonical exons and between donor and acceptor genes of trans-splicing shown in box plot. The bottom and top of the box represent the first and third quartiles, and the band inside the box represents the second quartile (the median). The whiskers extend 1.5 interquartile range (third quartile minus first quartile) upward from the third quartile, and downward from the first quartile. Outliers are marked with cross. The putative distance for DfChr is computed only for those sites on different scaffolds of unknown chromosomal location, assuming that donor and acceptor scaffolds are located on the same chromosome and that both genes are arranged in the same orientation, with the donor upstream of the acceptor. (c) Length of canonical transcripts, trans-spliced products and donor and acceptor segments of trans-splicing events shown in box plot. The parameters used in the box plots are the same as those of b. (d) Frequency distribution of transcripts with different number of exons. The x axis indicates the number of exons for each transcript types. Donor retained, trans-spliced transcript with retained donor segments; donor discarded, transcript with discarded donor segments; acceptor retained, trans-spliced transcript with retained acceptor segments; and acceptor discarded, transcript with discarded acceptor segments. (e) Nucleotide conservation at the junction sites of canonical and trans-spliced genes, shown with WebLogo(v3.3). The y axis indicates the relative frequency of each nucleic acid in bits (a unit of entropy). (f) Predicted proteins from intergenic trans-splicing events in insects. To qualify, translation must initiate within the donor segment and must contain at least ten amino acids in both the donor and acceptor segments.

Figure 3. Mod(mdg4)-like trans-splicing events conserved in insects.

(a) Two different types of mod(mdg4)-like trans-splicing. AA, number of amino acids; Red bar, donor transcript (5′-common exons); blue bar, acceptor transcript (3′-alternative exons). Arrows denote direction of transcripts. *Assuming that scaffold_278 and scaffold_162 are located on the same chromosome in P. xylostella. (b) Diagram of the mod(mdg4) locus in D. plexippus. Red bar, donor transcript (5′-common exons); blue bar, acceptor transcript (3′-alternative exons). Black arrows indicate strand direction. Mod(mdg4) isoforms validated by RT–PCR are marked by orange arrows. The RT–PCR is performed as described in Methods using mixed D. plexippus samples (n=3). The name of each acceptor is denoted by DPOGS20 (prefix) plus the four-digit label of blue bar(s).

Figure 4. Sequence diversification of mod(mdg4) products throughout evolution.

(a) Exon structure of the mod(mdg4) N-terminus. The BTB domain-encoding region is marked by a dashed box. Grey box, exon. (b) Sequence similarity of N-terminal regions. Number of conserved amino acid at each position is indicated by blue curve. Sequences are aligned based on the BTB domain model using HMMalign (see Methods for details). Only alignment of the conserved region is shown. Dme, D. melanogaster; Aae, A. aegypti; Aga, A. gambiae; Bmo, B. mori; Dpl, D. plexippus; Pxy, P. xylostella; Dpu, D. pulex. (c) Phylogenetic analysis of C-terminal FLYWCH domains using Maximum Likelihood analysis (see Methods for details). FLYWCH domains from different species are marked in distinct colours. The Dipteran and Lepidopteran groups are separated by different background colours. The same tree with detailed labelling is provided in Supplementary Fig. 5.

Figure 5. Examples of non-mod trans-splicing events.

(a) Comparison of BGIBMGA008294_exon4::SIBSBM001135_exon2 trans-splicing in silkworm and its homologues from other species. Exons retained in the trans-spliced product are enclosed in dashed boxes. Black block, coding exons; grey block, untranslated exons; red block, donor exons; blue block, acceptor exons. Cpi, C. pipiens; Aae, A. aegypti; Aga, A. gambiae; Dme, D. melanogaster; Pxy, P. xylostella; Bmo, B. mori; Hme, H. melpomene; Dpl, D. plexippus; Tca, T. castaneum; Ame, A. mellifera; Aec, A. echinatior; Cfl, C. floridanus; Api, A. pisum; Dpu, D. pulex. (b) Schematic diagram of trans-splicing between two paralogues, NM_079398 and NM_057620. Red denotes donor exons, and blue indicates acceptor exons. Sequence reads covering the junction site are aligned, with critical residues highlighted in background colours consistent with those of the donor or acceptor.