The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene:
The venoms of many animals, including marine cone snails, scorpions, snakes, sea anemones, spiders and platypus contain many peptides that are produced by post-translational processing of a larger precursor . The precursors of cone snail and sea anemone toxins contain both a signal sequence and a propeptide region, whereas the propeptide region is generally absent in scorpion and snake toxin precursors , . The situation is more complex in spiders, with propeptide regions typically present in transcripts encoding “short” toxins (<5 kDa) , , , ,  but absent from transcripts encoding longer toxins . However, in all cases, the precursor encodes only a single copy of the mature toxin sequence.
Since most venom research (not only on spiders) has been focused on the isolation and identification of peptides via proteomic and transcriptomic approaches, mainly with a view towards developing new pharmacological tools and therapeutic leads, there is a paucity of information on the number of toxin genes, their genetic architecture, and the genetic mechanisms underpinning toxin diversification. A small number of genes encoding venom peptides have been described from cone snails, scorpions, sea anemones and snakes and with only a few exceptions they all have an intro-exon architecture. In conotoxins, one intron is positioned between the signal and propeptide coding regions and another between the propeptide and mature toxin region , whereas a variable number of introns (1 to 3) can be found in snake-toxin genes , , . There are examples of both intronless and intron-containing venom genes in scorpions , –.
The gene encoding μ-diguetoxin-Dc1a from the araneomorph spider D. canities  was the first spider-venom peptide gene to be sequenced. This gene contains five introns that span 5.5 kb; the first two introns are located within the 5′ UTR and propeptide-encoding region while the other two are situated in the region encoding the mature toxin (Fig. 1A). More recently, 24 genes encoding ICK-containing venom peptides were sequenced from two mygalomorph spiders, namely the Chinese bird spider Haplopelma huwenum and the Chinese Black Earth Tiger tarantula Haplopelma hainanum–. All of these genes lacked introns, in striking contrast to the gene encoding μ-diguetoxin-Dc1a.
In this study we determined for the first time the architecture of genes encoding ICK-containing venom peptides from the highly venomous Australian funnel-web spider. All three genes we examined were intronless, including the gene encoding the lethal δ-hexatoxin-Hi1a peptide. Thus, to date, all of the 27 genes encoding ICK-containing venom-peptides that have been sequenced from primitive mygalomorph spiders are intronless, which raises interesting questions about the ancestral state of genes encoding spider-venom peptides as well as the mechanism of toxin diversification.
Spiders evolved from an arachnid ancestor more than 300 million years ago (Mya). Extant spiders are divided into the suborders Opisthothelae and Mesothelae, with the latter comprising a single family of primitive, venom-less burrowing spiders . Opisthothelae is further divided into two infraorders, Mygalomorphae and Araneomorphae, sometimes referred to as “primitive” and “modern” spiders, respectively . Molecular clock analyses suggest that mygalomorphs and araneomorphs split ~280 Mya , which is consistent with the fossil record. The earliest mygalomorph fossil dates to the early Triassic period ~240 Mya, while the earliest araneomorph fossils date to ~225 Mya . If the complex intron-exon architecture determined for μ-diguetoxin-Dc1a is representative of genes encoding araneomorph toxins then either introns were lost secondarily by mygalomorph spiders following their divergence from araneomorphs more than 250 Mya or the ancestral state of spider-venom peptide genes is intronless.
Minimization of genome size through extensive loss of ancestral introns has been reported for microsporidia, fungi, red algae, and apicomplexans . At this stage it is unclear whether there has been tendency towards minimization of genome size in mygalomorphs, but this seems unlikely. Recent surveys of ~120 araneomorph spiders ,  have revealed an enormous variation in genome size (700–5,500 Mb) but there are no current estimates for genome size in mygalomorph spiders. Loss of introns is associated with evolutionary diversification of snake-venom disintegrins but in this case intron loss is correlated with a corresponding reduction in protein size . Given the growing consensus that the ancestral bilaterian was rich in introns and that differences in intron numbers between animals largely reflect different levels of intron loss , the most parsimonious explanation of the current sparse data on spider-venom peptide genes is that ancestral spider-toxin genes contained introns (as seen in the gene encoding μ-diguetoxin-Dc1a) but these were lost at an early stage in the evolution of genes encoding ICK toxins. Since Australian funnel-web spiders (family Hexathelidae) and tarantulas (family Theraphosidae) diverged more than 200 Mya, mygalomorph spiders presumably dispensed with introns in genes encoding ICK toxins at a very early stage of venom evolution.
The implications of intron loss or gain in genes expressing spider-venom peptides will not be understood until more genes are sequenced from a greater diversity of spiders. However, the apparent widespread loss of introns in genes encoding mygalomorph ICK toxins raises questions about the mechanism of toxin diversification, as alternative splicing and other intron editing mechanisms are clearly not being used to expand the repertoire of venom peptides. Moreover, the absence of introns makes it difficult to invoke exon shuffling as the mechanism by which spiders created larger “double-knot toxins” comprised of two tandemly-repeated ICK domains .
The absence of introns suggests that the mechanism of diversification of spider-venom ICK toxins differs from that employed by venomous cone snails to expand their toxin repertoire. As for spider-venom ICK toxins, disulfide-rich peptides from cone snail venoms are initially produced as prepropeptides that are post-translationally processed to yield the mature toxin. These cone snail toxins are encoded by genes that architecturally resemble those encoding the spider-venom peptide μ-diguetoxin-Dc1a, with three exons separated by large (>1 kb) introns . Exon III, which encodes the mature toxin, appears to have evolved at a 10-fold higher rate than exon I, which encodes the signal peptide, and it has been suggested that the separation of these exons by much larger intronic sequences has facilitated their markedly different rates of mutation . Spider-venom ICK toxins show a similar disparity in the rate of mutation between the signal peptide and mature toxin , without the benefit of these regions being encoded by separate, widely separated exons. Hence, it will be interesting to determine what allows the mature-toxin region to be extensively mutated over evolutionary time while the signal peptide that is only ~40–60 bp upstream remains under strong negative selection pressure .
Most toxin genes are transcribed at high frequency during venom regeneration, and thus intronless genes might diversify over time due to elevated rates of mutation that are sometime associated with highly transcribed genes, a process known as transcription-associated mutation (TAM). TAM is associated with an increased frequency of mutations such as base replacements, deletions, and recombination . Other heavily transcribed intronless genes such as those involved in immune recognition and response diversify via mechanisms of recombination, somatic hypermutation, class switch recombination, and gene conversion , . Whether any of these processes underlie the diversification of intronless spider-venom genes remains to be seen.
In summary, we have shown that the gene encoding the lethal δ-hexatoxin-Hi1a peptide as well as the genes encoding two other ICK-containing toxins (ω-hexatoxin-Hi2a and U3-hexatoxin-Hi1a) from the Australian funnel-web spider Hadronyche infensa are intronless. This rules out alternative splicing as a mechanism for enhancing venom diversity and it raises still to be answered questions about the ancestral state of spider toxin genes.
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