Gene structure, regulatory control, and evolution of black widow venom latrotoxins

Abstract

Black widow venom contains α-latrotoxin, infamous for causing intense pain. Combining 33 kb of Latrodectus hesperus genomic DNA with RNA-Seq, we characterized the α-latrotoxin gene and discovered a paralog, 4.5 kb downstream. Both paralogs exhibit venom gland specific transcription, and may be regulated post-transcriptionally via musashi-like proteins. A 4 kb intron interrupts the α-latrotoxin coding sequence, while a 10 kb intron in the 3' UTR of the paralog may cause non-sense-mediated decay. Phylogenetic analysis confirms these divergent latrotoxins diversified through recent tandem gene duplications. Thus, latrotoxin genes have more complex structures, regulatory controls, and sequence diversity than previously proposed.

Keywords: Genomics; Latrodectus; Molecular evolution; Neurosecretion; Venom; α-Latrotoxin.

Figure 1

Mapping of RNA-Seq data to the 33 kb L. hesperus genomic reference sequence showing strong venom gland specific expression of tandem latrotoxin paralogs. Top ruler shows location in the reference sequence. Representative reads from the L. hesperus venom gland (top panel), cephalothorax (middle) and silk gland (bottom) RNA-seq libraries are shown mapped to the reference using Tophat with visualization in IGV. Reads mapping across intron boundaries are connected by thin lines and show location of splice junctions. At the bottom the gene structure of the two latrotoxins encoded by the fosmid sequence as determined by Cufflinks is diagrammed. In exonic regions, coding sequences are in gray, UTRs in black. The dashed line indicates the extended 3′ UTR region predicted by the Trinity assembled transcript. The position of the mariner element is indicated in the α-latrotoxin intron, and the RepeatMasker predicted retrotransposon positions are indicated in red in the intron of the downstream paralog.

Figure 2

MAKER-annotated L. hesperus genomic sequence illustrating latrotoxin introns containing transposable elements. The top annotation track shows Augustus predicted gene structure. The middle track shows alignments of RNA-Seq assembled transcripts and ESTs to the fosmid (Cufflinks assembled transcripts labeled with prefix “CUFF”, Trinity assembled transcripts labeled with prefix “venom_comp”, all other sequences are L. hesperus venom gland ESTs labeled with their GenBank Accession numbers), while the bottom section shows repeat regions identified by RepeatMasker (all features annotated in this track from 0–19 kb are simple repeat/low complexity regions; features between 22 kb–32 kb are retrotransposon fragments). The position of the putative Tc1/mariner family transposon not predicted by RepeatMasker is shown in green.

Figure 3

Evolutionary relationships of latrotoxin paralogs indicating the relatively recent duplication of tandem latrotoxins. Shown is a midpoint rooted Bayesian phylogenetic tree of latrotoxin proteins. Numbers at nodes indicate clade posterior probabilities (PP). The node marked by an asterisk has a PP of 1.00. Sequences from L. tredecimguttatus are in red and L. hesperus in blue; sequences from L. geometricus and S. grossa are in orange or green, respectively. Arrows indicate the tandem sequences assembled by Cufflinks from this study. All other L. hesperus and L. tredecimguttatus sequences were from de novo assembly of RNA-Seq reads. Shaded beige boxes label clades that contain a functionally characterized paralog. The larger gray shaded clade contains the two genomic insert latrotoxins and more closely related sequences, including all α-latrotoxins. Branches tested for sites under positive selection are indicated by the number 1 in purple text.