Alternative mRNA Splicing in Three Venom Families Underlying a Possible Production of Divergent Venom Proteins of the Habu Snake, Protobothrops flavoviridis

Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake

The habu genome reveals accelerated evolution of venom protein genes

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition

Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake

Protobothrops flavoviridis (Crotalinae) venom gland phospholipase A2 (PLA2) isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities in their products. For elucidation of the multiplication mechanism of PLA2 genes, a 25,026 bp genome segment harboring five PLA2 isozyme genes was obtained from Amami-Oshima P. flavoviridis liver and sequenced. The gene PfPLA 2 encoded [Lys49] LA2 called BPII, the gene PfPLA 4 neurotoxic [Asp49]PLA2 called PLA-N, the gene PfPLA 5 basic [Asp49]PLA2 called PLA-B, and PfPLA 1(Ï¿) and PfPLA 3(Ï¿) were the inactivated genes. The 5' truncated reverse transcriptase (RT) elements, whose intact forms constitute long interspersed nuclear elements (LINEs), were found in close proximity to the 3' end of PLA2 genes and named PLA2 gene-coupled RT fragments (PcRTFs). The facts that PcRTFs have the stemâ¿¿loop and repetitive sequence in the 3' untranslated region (UTR) which is characteristic of CR1 LINEs suggest that PcRTFs are the debris of P. flavoviridis ancestral CR1 LINEs, denoted as PfCR1s. Since the associated pairs of PLA2 genes and PcRTFs are arranged in tandem in the 25,026 bp segment, it is thought that an ancestral PLA2 gene-PfCR1 unit (PfPLA-PfCR1) which was produced by retrotransposition of PfCR1 by itself to the 3' end of PLA2 gene duplicated several times to form a multimer of PfPLA-PfCR1, a cluster of PLA2 genes, in the period after Crotalinae and Viperinae snakes branched off. Recombinational hot spot of a 37 bp segment, named Scomb, was found in the region 548 bp upstream from the TATA box of PLA2 genes. Thus, it could be assumed that multiplication of PfPLA-PfCR1 occurred by unequal crossing over of the segment, -Scomb-PfPLA-PfCR1-Scomb-. The PfCR1 moieties were afterward disrupted in the 5' portion to PcRTFs. The detection of two types of PcRTFs different in length which were produced by elimination of two definitive sequences in PfCR1 moiety possibly by gene conversion clearly supports such process but not multiplication of the PLA2 gene-PcRTF unit

The Finding of a Group IIE Phospholipase A2 Gene in a Specified Segment of Protobothrops flavoviridis Genome and Its Possible Evolutionary Relationship to Group IIA Phospholipase A2 Genes

The genes encoding group IIE phospholipase A2, abbreviated as IIE PLA2, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA2s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA2s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA2 gene. Moreover, a group IIA PLA2 gene was found in the 5' upstream of IIE PLA2 gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA2 gene, IIE PLA2 gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA2s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA2 isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA2 genes may be evolutionarily derived from the IIE PLA2 gene