RESUMEN
Studying animal toxin evolution requires sequences of these proteins and peptides, and transcript sequences allow for the construction of cladograms and evaluation of selection pressures from nonsynonymous and synonymous nucleotide mutation ratios. In addition, these translated sequences can be useful as custom databases for peptide identifications within venoms and for better proteomic quantification. Obtaining these transcripts is achieved by sequencing cDNA originating from venom gland tissue or venom. This chapter provides the methodology for (1) targeted sequencing of transcripts from a single venom protein family (RNA isolation and 3'RACE [rapid amplification of cDNA ends]), (2) generation of a venom gland transcriptome with next-generation sequencing (NGS) technology (de novo transcriptome assembly, toxin transcript identification, quantification, and positive selection analysis), and (3) combined high-throughput proteomics to identify secreted venom components. Transcriptomics has become fundamental for studying toxin evolution, but it creates many challenges for scientists who are unfamiliar with working with RNA, managing large NGS datasets and executing the required programs, particularly considering that there is an overabundance of available software in this field and not all perform optimally for venom gland transcriptome assembly. This chapter provides one pipeline for the integration of both low- and high-throughput transcriptomics with proteomics to characterize venoms.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteómica/métodos , Animales , Biología Computacional/métodos , Transcriptoma/genética , Ponzoñas/análisisRESUMEN
The Harderian gland is a cephalic structure, widely distributed among vertebrates. In snakes, the Harderian gland is anatomically connected to the vomeronasal organ via the nasolacrimal duct, and in some species can be larger than the eyes. The function of the Harderian gland remains elusive, but it has been proposed to play a role in the production of saliva, pheromones, thermoregulatory lipids and growth factors, among others. Here, we have profiled the transcriptomes of the Harderian glands of three non-front-fanged colubroid snakes from Cuba: Caraiba andreae (Cuban Lesser Racer); Cubophis cantherigerus (Cuban Racer); and Tretanorhinus variabilis (Caribbean Water Snake), using Illumina HiSeq2000 100â¯bp paired-end. In addition to ribosomal and non-characterized proteins, the most abundant transcripts encode putative transport/binding, lipocalin/lipocalin-like, and bactericidal/permeability-increasing-like proteins. Transcripts coding for putative canonical toxins described in venomous snakes were also identified. This transcriptional profile suggests a more complex function than previously recognized for this enigmatic organ.
Asunto(s)
Colubridae/metabolismo , Regulación de la Expresión Génica/fisiología , Glándula de Harder/metabolismo , Proteínas de Reptiles/biosíntesis , Venenos de Serpiente/biosíntesis , Transcriptoma/fisiología , Animales , Colubridae/genética , Cuba , Proteínas de Reptiles/genética , Venenos de Serpiente/genéticaRESUMEN
With an extensive range in tropical and subtropical waters of Indo-Australian region and the Pacific coast of the American continent, the yellow-bellied sea snake, Hydrophis platurus, is the most broadly distributed snake species on our planet. We report a comparative analysis of the mRNA and microRNA transcriptional profiles of the venom glands of Costa Rican juvenile and adult yellow-bellied sea snakes, and correlate these datasets with the previously reported venom proteome gathered from an adult specimen of the same population of Playa del Coco. The transcriptomic profiles are qualitatively and quantitatively remarkably similar across both age classes, being overwhelmingly (>99%) dominated by only three toxin classes, three-finger toxins (3FTx, 88%), phospholipases A2 (PLA2, 10-11%), and cysteine-rich secretory proteins (CRISP, 1%). The only appreciable difference between the transcriptomes of juvenile and adult Costa Rican yellow-bellied sea snake lies in the relative abundance of short and long neurotoxic 3FTxs. Comparison of venom gland transcriptome and venom proteome datasets suggest that the venom arsenal of adult H. platurus from Playa del Coco (CR) is subjected to post-transcriptional modulation, involving repression of the translation of pelamitoxin a-encoding mRNAs and enhancement of the translational activity of PLA2 and CRISP mRNAs.
Asunto(s)
Venenos Elapídicos/metabolismo , Hydrophiidae/genética , Transcriptoma , Factores de Edad , Animales , Costa Rica , Hydrophiidae/metabolismo , MicroARNs , Proteoma , ARN Mensajero , Glándulas Salivales/metabolismoRESUMEN
Adult rattlesnakes within genus Crotalus express one of two distinct venom phenotypes, type I (hemorrhagic) and type II (neurotoxic). In Costa Rican Central American rattlesnake, ontogenetic changes in the concentration of miRNAs modulate venom type II to type I transition. Venomics and venom gland transcriptome analyses showed that adult C. simus and C. tzabcan expressed intermediate patterns between type II and type I venoms, whereas C. culminatus had a canonical type I venom. Neonate/juvenile and adult Mexican rattlesnakes showed notable inter- and intraspecific variability in the number, type, abundance and ontogenetic shifts of the transcriptional and translational venom gland activities. These results support a role for miRNAs in the ontogenetic venom compositional changes in the three congeneric Mexican rattlesnakes. It is worth noting the finding of dual-action miRNAs, which silence the translation of neurotoxic heterodimeric PLA2 crotoxin and acidic PLA2 mRNAs while simultaneously up-regulating SVMP-targeting mRNAs. Dual transcriptional regulation potentially explains the existence of mutually exclusive crotoxin-rich (type-II) and SVMP-rich (type-I) venom phenotypic dichotomy among rattlesnakes. Our results support the hypothesis that alterations of the distribution of miRNAs, modulating the translational activity of venom gland toxin-encoding mRNAs in response to an external cue, may contribute to the mechanism generating adaptive venom variability.
Asunto(s)
Venenos de Crotálidos/genética , Crotalus/genética , MicroARNs/genética , Proteogenómica/métodos , Proteoma/genética , Transcriptoma , Factores de Edad , Animales , Secuencia de Bases , Cromatografía de Fase Inversa/métodos , Venenos de Crotálidos/biosíntesis , Venenos de Crotálidos/clasificación , Venenos de Crotálidos/aislamiento & purificación , Crotalus/crecimiento & desarrollo , Crotalus/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Variación Genética , MicroARNs/metabolismo , Anotación de Secuencia Molecular , Biosíntesis de Proteínas , Proteogenómica/instrumentación , Proteoma/metabolismo , Especificidad de la EspecieRESUMEN
Bothriechis is a genus of eleven currently recognized slender and arboreal venomous snakes, commonly called palm-pitvipers that range from southern Mexico to northern South America. Despite dietary studies suggesting that palm-pitvipers are generalists with an ontogenetic shift toward endothermic prey, venom proteomic analyses have revealed remarkable divergence between the venoms of the Costa Rican species, B. lateralis, B. schlegelii, B. supraciliaris, and B. nigroviridis. To achieve a more complete picture of the venomic landscape across Bothriechis, the venom proteomes of biodiversity of the northern Middle American highland palm-pitvipers, B. thalassinus, B. aurifer, and B. bicolor from Guatemala, B. marchi from Honduras, and neonate Costa Rican B. lateralis and B. schlegelii, were investigated. B. thalassinus and B. aurifer venoms are comprised by similar toxin arsenals dominated by SVMPs (33-39% of the venom proteome), CTLs (11-16%), BPP-like molecules (10-13%), and CRISPs (5-10%), and are characterized by the absence of PLA2 proteins. Conversely, the predominant (35%) components of B. bicolor are D49-PLA2 molecules. The venom proteome of B. marchi is similar to B. aurifer and B. thalassinus in that it is rich in SVMPs and BPPs, but also contains appreciable amounts (14.3%) of PLA2s. The major toxin family found in the venoms of both neonate B. lateralis and B. schlegelii, is serine proteinase (SVSP), comprising about 20% of their toxin arsenals. The venom of neonate B. schlegelii is the only palm-pitviper venom where relative high amounts of Kunitz-type (6.3%) and γPLA2 (5.2%) inhibitors have been identified. Despite notable differences between their proteomes, neonate venoms are more similar to each other than to adults of their respective species. However, the ontogenetic changes taking place in the venom of B. lateralis strongly differ from those that occur in the venom of B. schlegelii. Thus, the ontogenetic change in B. lateralis produces a SVMP-rich venom, whereas in B. schlegelii the age-dependent compositional shift generates a PLA2-rich venom. Overall, genus-wide venomics illustrate the high evolvability of palm-pitviper venoms. The integration of the pattern of venom variation across Bothriechis into a phylogenetic and biogeographic framework may lay the foundation for assessing, in future studies, the evolutionary path that led to the present-day variability of the venoms of palm-pitvipers. SIGNIFICANCE: Bothriechis represents a monophyletic basal genus of eleven arboreal palm-pitvipers that range from southern Mexico to northern South America. Despite palm-pitvipers' putative status as diet generalists, previous proteomic analyses have revealed remarkable divergence between the venoms of Costa Rican species, B. lateralis, B. schlegelii, B. supraciliaris, and B. nigroviridis. Our current proteomic study of Guatemalan species, B. thalassinus, B. aurifer, and B. bicolor, Honduran B. marchi, and neonate B. lateralis and B. schlegelii from Costa Rica was undertaken to deepen our understanding of the evolutionary pattern of venom proteome diversity across Bothriechis. Ancestral characters are often, but not always, preserved in an organism's development. Venoms of neonate B. lateralis and B. schlegelii are more similar to each other than to adults of their respective species, suggesting that the high evolvability of palm-pitviper venoms may represent an inherent feature of Bothriechis common ancestor. Our genus-wide data identified four nodes of venom phenotype differentiation across the phylogeny of Bothriechis. Integrated into a phylogenetic and biogeographic framework, the pattern of venom variation across Bothriechis may lay the groundwork to establish whether divergence was driven by selection for efficient resource exploitation in arboreal 'islands', thereby contributing to the ecological speciation of the genus.
Asunto(s)
Biodiversidad , Venenos de Crotálidos/química , Proteoma/análisis , Factores de Edad , Animales , Evolución Biológica , Venenos de Crotálidos/enzimología , Fosfolipasas A2/análisis , Filogenia , Proteómica/métodos , Serina Proteasas/análisis , Toxinas Biológicas/análisis , ViperidaeRESUMEN
Over the last three decades, transcriptomic studies of venom gland cells have continuously evolved, opening up new possibilities for exploring the molecular diversity of animal venoms, a prerequisite for the discovery of new drug candidates and molecular phylogenetics. The molecular complexity of animal venoms is much greater than initially thought. In this review, we describe the different technologies available for transcriptomic studies of venom, from the original individual cloning approaches to the more recent global Next Generation Sequencing strategies. Our understanding of animal venoms is evolving, with the discovery of complex and diverse bio-optimized cocktails of compounds, including mostly peptides and proteins, which are now beginning to be studied by academic and industrial researchers.
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Transcriptoma , Ponzoñas/metabolismo , Animales , Descubrimiento de Drogas , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Dolor/tratamiento farmacológico , Ponzoñas/uso terapéuticoRESUMEN
Bothriechis is considered a monophyletic, basal genus of arboreal Neotropical pitvipers distributed across Middle America. The four species found in Costa Rica (B. lateralis, B. schlegeli, B. nigroviridis, B. supraciliaris) differ in their venom proteomic profiles, suggesting that different Bothriechis taxa have evolved diverse trophic strategies. In this study, we isolated a phospholipase A2 (PLA2) from B. lateralis venom, aiming at increasing our knowledge on the structural and functional characteristics of group II acidic PLA2s, whose toxic actions are generally more restricted than those displayed by basic PLA2s. The new acidic enzyme, BlatPLA2, occurs as a monomer of 13,917 Da, in contrast to many basic group II PLA2s which associate into dimers and often display myotoxicity and/or neurotoxicity. Its amino acid sequence of 122 residues predicts an isoelectric point of 4.7, and displays significant differences with previously characterized acidic PLA2s, with which it shows a maximum sequence identity of 78%. BlatPLA2 is catalytically active but appears to be devoid of major toxic activities, lacking intravenous or intracerebroventricular lethality, myotoxicity, in vitro anticoagulant activity, and platelet aggregation or inhibition effects. Phylogenetic relationships with similar group II enzymes suggest that BlatPLA2 may represent a basal sequence to other acidic PLA2s. Due to the metabolic cost of venom protein synthesis, the presence of a relatively abundant (9%) but non-toxic component is somewhat puzzling. Nevertheless, we hypothesize that BlatPLA2 could have a role in the pre-digestion of prey, possibly having retained characteristics of ancestral PLA2s without evolving towards potent toxicity.
Asunto(s)
Venenos de Crotálidos/enzimología , Fosfolipasas A2/genética , Viperidae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Análisis por Conglomerados , Biología Computacional , Costa Rica , Reacciones Cruzadas/efectos de los fármacos , Venenos de Crotálidos/genética , Edema/inducido químicamente , Punto Isoeléctrico , Ratones , Datos de Secuencia Molecular , Fosfolipasas A2/química , Fosfolipasas A2/toxicidad , Filogenia , Agregación Plaquetaria/efectos de los fármacos , Análisis de Secuencia de ADN , Homología de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Pruebas de ToxicidadRESUMEN
BACKGROUND: Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. RESULTS: Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a ß-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9-18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulthood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. CONCLUSIONS: Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112-125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.
Asunto(s)
Venenos de Crotálidos/química , Crotalus/genética , MicroARNs/genética , Proteómica , Animales , Animales Recién Nacidos , Crotalus/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Fenotipo , Retroelementos/genética , Factores de Tiempo , TranscriptomaRESUMEN
BACKGROUND: A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. RESULTS: The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani. CONCLUSIONS: Our comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.
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Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Glándulas Salivales/metabolismo , Análisis de Secuencia de ADN/métodos , Venenos de Serpiente/genética , Serpientes/genética , Animales , Costa Rica , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serpientes/clasificación , Serpientes/metabolismoRESUMEN
The Mex67p protein, together with Mtr2p, functions as the mRNA export receptor in Saccharomyces cerevisiae by interacting with both mRNA and nuclear pore complexes. To identify genes that interact functionally with MEX67, we used transposon insertion to search for mutations that suppressed the temperature-sensitive mex67-5 allele. Four suppressors are described here. The screen revealed that mutant Mex67-5p, but not wild-type Mex67p, is a target of the nuclear protein quality control mediated by San1p, a ubiquitin-protein ligase that participates in degradation of aberrant chromatin-associated proteins. Our finding that overexpression of the SPT6 gene alleviates the growth defects of the mex67-5 strain, together with the impairment of poly(A)(+) RNA export caused by depletion of Spt6p or the related protein Iws1p/Spn1p, supports the mechanism proposed in mammalian cells for Spt6-mediated co-transcriptional loading of mRNA export factors during transcription elongation. Finally, our results also uncovered genetic connections between Mex67p and the poly(A) nuclease complex and with components of chromatin boundary elements.