Integrated “omics” profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus simus
© Durban et al.; licensee BioMed Central Ltd. 2013
Received: 12 November 2012
Accepted: 14 March 2013
Published: 10 April 2013
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.
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 β-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 adulhood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus.
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.
KeywordsOntogenetic venom shift Venomics Snake venom gland transcriptomics 454 pyrosequencing Ion-Torrent microRNA profiling Crotalus simus simus
The presence of a venom system is a shared derived character of the advanced snakes [1–3]. Venom represents an adaptive trophic trait in snake evolution [4–7] that has played a central role in the origin of the advanced snakes during the Cenozoic era [8, 9]. The diversity of modern snakes appeared during the Paleocene period of the Cenozoic Era, approximately 54–64 Mya, following the split of the Pareatidae from the remaining Caenophidians . In Occidental culture, Francesco Redi (1626–1697), court physician to Ferdinando II de’ Medici, Grand Duke of Tuscany and his successor, Cosimo III, is credited for discovering how vipers produce venom and inject it into their prey . One century latter, Abbé Gasparo Ferdinando Felice Fontana (1730–1805), first director of the Museum of Physics and Natural History in Florence, performed experiments on the venom of the European viper. His classic text published in 1781  is regarded the Opera Prima of modern toxinology .
Research on venoms has been continuously advanced by technological developments . Particularly for the past decade, and fueled by the application of "omic" technologies [14–18], the field of molecular toxinology has experienced a sustained exponential growth. In-depth venom proteomics and transcriptomic analyses have recently become available for a number of venomous snake lineages, revealing a vast unexplored source of evolutionary innovation [2, 19, 20]. Snake venoms are well documented as having different venom compositions and toxicity based on taxonomic or geographical locations . Inter- and intraspecific individual, gender-specific, regional, and seasonal variations in venom toxin composition may reflect local adaptations conferring fitness advantages to individuals specializing on different prey, or phylogenetic carry on. Understanding the processes that drive the evolution of snake venom variability is a topic of intense research interest in molecular and evolutionary toxinology. Recent studies of the molecular basis of adaptations have sought to understand the relative importance of gene regulation effects as determinants of venom phenotype [6, 22–24]. Most significantly, a number of snakes show age-related (ontogenetic) changes in venom composition [25–32]. Surprisingly, despite previous suggestions that ontogenetic changes in venom are prey-related , juvenile Dusky Pigmy rattlesnakes, Sistrurus miliarius barbouri, raised on different diets showed similar albeit highly-variable venom compositions by the end of a 26-months study, suggesting little effect of diet on the overall make-up of venom in snakes this age or younger . Over the same period shifts in venom composition occurred in females raised on different prey in all diet treatments with the magnitude of those changes strongly related to diet. This work provided evidence that venom composition is somewhat plastic in both juvenile and adult S. m. barbouri and that, at least in adults, prey consumed may influence the relative abundance of possibly functionally-distinct classes of venom proteins . However, the molecular mechanisms that underly age-related changes in venom remain elusive.
In this work 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. Biogeographical data indicate a basal cladogenesis in the Central American C. simus clade, dating back to the late Miocene/early Pliocene (6.4-6.7 Mya) . Neonate and juvenile C. s. simus venoms contain large relative amounts of crotoxin, a heterodimeric PLA2 molecule exhibiting presynaptic β-neurotoxicity, along with low concentration of hemorrhagic snake venom metalloproteinases (SVMPs) . By contrast, adult C. s. simus venom presents a high content of SVMP and is largely devoid of neurotoxic activity [29, 35, 36]. Juvenile and adult C. s. simus venom phenotypes broadly correspond, respectively, to type II (low metalloproteinase activity and high toxicity, LD50 <1 μg/g mouse body weight) and type I (high levels of SVMPs and low toxicity, LD50 >1 μg/g mouse body weight) venoms defined by Mackessy [37, 38]. Here we investigate the transition from type II to type I venom phenotype in C. s. simus through an integrated "omics" approach involving proteomic survey of time-course venom composition variation, from birth through adulthood, and Next Generation sequencing of the venom gland transcriptomes and microRNAs of neonate and adult specimens. With this experimental design we intended to determine whether venom gene expression is transcriptionally controlled by developmental stage-dependent factors, or whether the ontogenetic changes in venom composition involve regulation at the post-transcriptional level.
Results and discussion
Time-resolved proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus
A previous proteomic survey of the venom of the Central American rattlesnake, C. s. simus, laid the foundation for understanding the changes in toxin composition and overall pharmacological features between adult (predominantly hemorrhagic) and newborn (mainly neurotoxic) snakes . This study revealed prominent stage-dependent protein expression changes between the pooled venoms from newborn and adult specimens from Costa Rica, characterized by a shift from a type II to a type I venom phenotype . Such a conspicuous venom composition change has been hypothesized to be related to variations in the size of prey consumed by snakes of different ages and the variable requirements for immobilizing and digesting them [37, 38]. It has also been suggested that the significance of venom adaptation to specific diets represents a trade-off between the metabolic cost of venom production and increasing foraging efficiency [7, 39]. In the present work we have sought to establish the molecular mechanism responsible for the reported ontogenetic shift in venom protein composition.
Analysis of the venom gland transcriptomes of neonate and adult C. s. simus. Retroelements
454 sequencing statistics
Mean length read (nt)
Max. length read (nt)
Assembled reads (%)
Number of contigs
Mean length contig (nt)
Max. length contig (nt)
Contig sequences were inspected for repetitive elements using Repeat Masker. 97,204 bases (1.93% of adult C. s. simus venom gland transcriptome) and 65,742 nucleotides (1.94% of the newborn venom gland transcriptome) were masked with N characters, a large part of them comprising Short and Long INterspersed repetitive Elements (SINEs and LINEs) retroelements (Additional file 1: Tables S1 and S2). Transposable elements (TE) that propagate within the host genome via RNA intermediates occupy a large fraction of eukaryotic genomes. Their mobility and amplification represent a major source of genomic variation [50, 51]. Retrotransposable elements have been reported in the transcriptomes of Bothrops insularis (4.1% of ESTs) , Lachesis muta (0.3%) , and Philodryas olfersii (4.1%) , in PLA2 genes from the venom gland of Vipera ammodytes[55, 56] and Protobothrops flavoviridis[57, 58], and in an E. ocellatus PIII-SVMP gene . Although their functional relevance in the venom gland remains unknown, transposable elements appear to be overrepresented in UTRs of mRNAs of rapidly evolving genes , suggesting that they have played a role in the diversification and expansion of these gene families [61, 62].
Sauria SINE have been characterized in all major lineages of squamate reptiles [62, 63], and phylogenetic analysis of E. ocellatus Sauria SINEs  indicated that their origin correlates with the time frame of the evolution of the snake venom system. Sauria SINEs may have arisen by a fusion of a tRNA-related sequence with the 3' end of a LINE  more than 200 million years ago and are uniquely widespread in lepidosaurian genomes . SINEs have no protein coding capacity, and their retrotransposition depends on a "target-primed reverse transcription" by autonomous partner LINEs, that encode an endonuclease for cleaving the genomic integration site and a reverse transcriptase to copy the RNA to DNA [65, 66]. Since Sauria SINEs share an identical 3' sequence with Bov-B LINEs, it has been proposed  that they utilize in trans the enzymatic machinery of Bov-B LINEs for their mobility and dispersal throughout the genome. In squamates and turtles, CR1 and L2 LINES are also partners of diverse SINEs [63, 67].
An overview of the landscape of retrotransposable elements reported in the sauropsida taxon, a sister group of mammals that includes all extant reptiles and birds, has recently emerged from analysis of the draft genomes of the red jungle fowl, Gallus gallus, and the green anole, Anolis carolinensis. Whereas a single type of LINE, CR1, comprises over 80% of all interspersed repeats in the chicken genome (200,000 copies; 9% of the chicken genome), approximately 30% of the A. carolinensis genome is composed of a wide diversity of LINE and SINE mobile element families. Since SINEs are among the largest multigene families in reptilian genomes, they may act as sites for homologous recombination events between dispersed SINEs, resulting in a variety of genetic rearrangements, including duplication, deletion and translocation, that likely represent mechanisms that generates genetic diversity in reptilian genomes . The prevalence of transposable elements in untranslated regions of mRNAs of recently expanded gene classes suggested that TEs could affect gene expression through the donation of transcriptional regulatory signals . The indistinguishable distribution of TEs in the venom gland transcriptomes of neonate and adult C. s. simus (Additional file 1: Tables S1 and S2) argues against this type of transcriptional regulation to explain the ontogenetic shift in venom composition.
Comparison of toxin-coding transcript distribution in newborn and adult C. s. simustranscriptomes provides clues for streamlining their divergent venom phenotypes
RPKM (Reads per Kilobase per Million mapped reads)-normalized contigs and singletons from the venom gland transcriptomes of newborn and adult C. s. simus aligned to a reference snake venom toxin ORF
Estimation of minimum number of toxin genes transcribed into the venom gland transcriptomes of newborn (N) and adult (A) C. s. simus
N + A
Distinct venom gland miRNA sets in newborn and adult snakes: modulators of the Central American rattlesnake's ontogenetic venom composition variation?
A. Summary of candidate miRNA sequencing statistics. B. Clustering and miRBase annotation statistics
Total raw reads
Prinseq quality filtered reads
FastX too-short reads
FastX adapter-only reads
Mean length read (nt)
BLAST miRBase hits
% Anolis hits
Concluding remarks and perspectives
The most important concept that emerges from our results is the possibility that miRNAs play a role in both the ontogenetic shift observed in certain venoms and in the plasticity of venoms from adult snakes underlying adaptations to changing environments. An important caveat of our current understanding of miRNA target recognition and post-transcriptional venom gland transcriptome regulation is the absence of mapeable snake genomes. Nonetheless, almost half of the most abundant miRNAs sequenced from the venom gland of both, newborn and adult, C. s. simus were orthologous to sequences in A. carolinensis. More significant is the fact that a C. s. simus newborn venom gland-exclusive miRNA and a venom gland miRNA uniquely found in adult snakes target, respectively, mRNAs encoding a PIII-SVMP and the B-subunit of crotoxin/Mojave toxin. Relevant to this point, Mojave toxin is a neurotoxic heterodimeric PLA2, whose translation into the venom proteome is ontogenetically regulated  (Figure 1). The noncovalent association of two dissimilar subunits, the small acidic, nonenzymatic, and nontoxic A-subunit with the basic and weakly toxic PLA2 B-subunit increases the lethal potency of the uncomplexed crotoxin B-subunit by enabling the toxin to reach its specific site of action at the neuromuscular junction [40, 41, 89–91]. Hence, miRNAs targeting a crotoxin subunit messenger would serve the goal of eliminating the generation of the neurotoxic heterodimer. 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 age-dependent proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus illustrated in Figure 1.
Large-scale proteomic analysis, performed for one miRNA (miR-223) in only one cell type (murine neutrophils), revealed that although some proteins were repressed by 50-80%, miR-223 typically had more modest effects , suggesting that perhaps other miRNAs in their endogenous context have targets for which protein output is more dramatically repressed. Clearly, a challenge ahead in molecular toxinology is to design laboratory experiments to uncover the impact, molecular details, and evolution of the regulatory miRNA-target interactions that shape venom phenotype during snake development. In this regard, it has been proposed that a single origin of venom in Squamata, the order of reptiles including lizards and snakes, dates back roughly 200 Mya to the Late Triassic/Early Jurassic period [1, 2, 93]. Existing snake venom toxins are the result of recruitment events 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 Mya of snake evolution. Given the central role that diet has played in the adaptive radiation of snakes , venom thus represents a key adaptation that has played an important role in the diversification of snakes. Our findings here reported support the view that understanding the basis for the phenotypic diversity of snake venoms requires a deep understanding of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results, though restricted to individual specimens from a single species, 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 and impact of these miRNAs on patterns of expression in the snake's venom gland, are further challenges for future research.
Proteomics assessment of the ontogenetic shift of venom composition in C. simus
Venoms from a large number (>25) of adult Crotalus s. simus specimens and from 11 captive-born siblings (from an age of 8-weeks to 21-months) kept at the serpentarium of the Instituto Clodomiro Picado, University of Costa Rica in San José were collected by snake biting on a parafilm-wrapped jar (adults and juveniles) or by aspiration from the fangs of neonates using an Eppendorf pipette. Crude venoms were centrifuged at low speed to remove cells and debris, lyophilized, weighed on a microbalance, and stored at −20°C until used. Venom proteins were separated by reverse-phase HPLC using a Teknokroma Europa C18 (0.4 cm × 25 cm, 5 mm particle size, 300 Å pore size) column and an Agilent LC 1100 High Pressure Gradient System equipped with DAD detector and micro-Auto-sampler. The flow-rate was set to 1 ml/min and the column was developed with a linear gradient of 0.1% TFA in water (solution A) and acetonitrile (solution B), isocratically (5% B) for 10 min, followed by 5–25 % B for 20 min, 25-45% B for 120 min, and 45-70% for 40 min. Isolated proteins were characterized as described .
Snake venom gland cDNA synthesis and 454 sequencing
Venom glands of an 8-week-old and an adult C. s. simus specimens were removed 3 days after venom milking, when transcription is maximal , from anesthetized snakes using fine forceps and immediately placed in RNAlater™ solution (Qiagen). About 50 mg of tissue were disrupted and homogenized by a rotor-stator homogenizer, and total RNA was isolated using RNeasy Mini kit (Qiagen), quantified in a NanoDrop ND_spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) through the A260/A280 ratio, and quality-checked on an agarose gel discerning the 28S and 18S bands of ribosomal RNA. First strand cDNA was synthesized using RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas), which selectively transcribes full-length polyadenylated mRNA. The manufacturer's recommendations were followed except where specified. Approximately 5 μg of total RNA was used as starting material. In order to avoid polymerase slippage, a modified 3' 54-mer adaptor (5' GAGCTAGTTCTGGAG(T)16VN), which includes a type IIs enzyme (GsuI) site (underlined), was used for first-strand synthesis. This modified oligonucleotide effectively converts the long run of adenosine residues at the polyA tail into a sequence that causes fewer problems for dideoxy sequencing chemistry, and thus the resulting cDNA libraries were enriched in 3'-end-transcripts. To avoid internal cuts, the cDNA was hemimethylated by adding 5-methyl-dCTP to the dNTPs mix. The first strand cDNA was used as template for second strand synthesis by E. coli DNA Polymerase I and RNase H. Double strand (ds) cDNA was precipitated with ethanol and the pellet was resuspended in 70 μL of nuclease-free water and subjected to enzymatic digestion with GsuI for 4 hours at 30°C. The enzyme was then inactivated at 65°C for 20 minutes and the digested cDNA was stored at −20°C. For 454 pyrosequencing, the GS FLX General DNA Library Preparation Method Manual workflow (Roche Diagnostics) was followed. To this end, 3 μg of final non-normalized cDNA library were sheared by nebulization into small fragments. The fragment ends were polished and short A/B adaptors were ligated onto both ends, providing priming regions to support both emulsion amplification and the pyrosequencing process. A biotin tag on the B adaptor allowed immobilization of the dscDNA library fragments onto streptavidin-conjugated magnetic beads and the subsequent isolation of the library of single strand cDNA sequencing templates. Each of the eight cDNA libraries was tagged with a unique 10-base sequence (MID, Multiplex IDentifier) that is recognized by the sequencing analysis software, allowing for automated sorting of MID-containing reads. Barcoded libraries were simultaneously sequenced in a Genome sequencing FLX Titanium System (Roche Applied Science) at Life Sequencing S.L. (Parc Científic Universitat de Valencia, Paterna, Valencia, Spain; http://www.lifesequencing.com) using the method developed by Margulies et al. .
Bioinformatic analysis of the 454 reads
454 data were analyzed using the workflow developed in  to identify sequences of toxin molecules by similarity search against nucleotide databases, which includes available NGS algorithms and in-house scripts. An initial quality test step of both the raw reads provided by the Genome Sequencer FLX System prior to the assembly process and the longer contig sequences obtained after running the 454 Newbler assembler program (version 2.6) (Titanium chemistry) was run using the Prinseq program (standalone version 0.17.4) . Interspersed repeats and low complexity DNA sequences were masked from the transcript reads using RepeatMasker (version 3.2.9) . RepeatMasker is available from the Institute for Systems Biology (http://www.systemsbiology.org) addressing http://repeatmasker.org. The program screens DNA sequences for interspersed repeats and low complexity DNA sequences included in the Repbase database (http://www.girinst.org). Repbase, a comprehensive database of repetitive element consensus sequences (update of 20 September 2011), operates as a service of the Genetic Information Research Institute (http://www.girinst.org). Data and computational resources for the Pre-Masked Genomes page is provided courtesy of the UCSC Genome Bioinformatics group (http://genome.ucsc.edu). Masked contig sequences were translated into the 6 possible reading frames and blasted against the non-redundant NCBI database (http://blast.ncbi.nlm.nih.gov, release of February 2012) and the UniProtKB/Swiss-Prot Toxin Annotation Program database (http://us.expasy.org/sprot/tox-prot), using BlastX and BlastN  algorithms (version 2.2.24), specifying a cut-off value of e-03 and BLOSUM62 as scoring matrix. Snake venom gland-specific transcripts were selected from best BLAST-hit descriptions identifying GenBank entries belonging to the taxonomic suborder Serpentes. This taxonomic group is represented by 44,141 nucleotide records comprising entries from 2,396 different species. A second filtering round was carried out using a list of keywords (including the acronyms of all known toxin protein families described so far to distinguish putative snake venom toxins from non-toxin (ribosomal, mitochondrial, nuclear, etc.) ordinary proteins . The phylogenetically nearest top-hit full-length sequence was designated as the reference sequence onto which all toxin family-specific reads were aligned to create a multiple alignment using COBALT . The multiple alignment was then parsed to create an assembled (consensus) toxin sequence in which each amino acid position is supported by at least four reads. We considered two contigs as different if their nucleotide sequences depart in more number of positions than expected from a sequencing error rate of 1.5%, and the same mutated residues were found in at least two other reads. Positions where two or more amino-acids fulfilled this criterium were annotated as variable residues suggesting the occurrence of different alleles (isoforms) of the protein.
Possible differential expression of venom proteins between adult and the newborn individuals was assessed with the non-parametric NOISeq-sim algorithm  using the following parameters recommended for counts without replicates in the NOISeq-sim manual were used: k (counts equal to zero) = 0.5; nss (number of samples to be simulated) ≥ 5; pnr (percentage of the total sequencing depth) = 0.2; v (variability in the total sequencing depth of the simulated sample) = 0.02. Reads were normalized by the length of the phylogenetically nearest sequence and a threshold of 0.9 was allowed.
Comparison of newborn and adult C. s. simusvenom transcriptomes
To assess the degree of similarity between transcripts synthesized by newborn an adult venom glands, a Perl script was written that aligned singletons and Newbler-assembled contigs onto the open reading frame (ORF) of the phylogenetically nearest protein sequence used as reference. The aligned nucleotide sequences were re-assembled with MIRA (http://www.chevreux.org/projects_mira.html) to infer the minimum number of different assemblies. These resulting sequences from newborn and adult individuals were compiled into a single FASTA file, translated into protein sequences, and manually inspected to discard possible mispaired BLAST annotations of local regions due to incorrect frame translations. Protein sequences were then clustered with CD-HIT (standalone version 4.5.7 built on 27th February 2012) [102, 103] to identify protein sequences shared between newborn and adult transcriptomes.
Snake venom gland microRNA profiling
Total RNA from neonate and adult venom glands was used to isolate small RNA libraries using a RNeasy Mini Kit following the manufacture's (Qiagen) instructions. Samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) through the 260/280 absorbance relation. 5 microliter of neonate (258.6 ng/microliter) and adult (490.7 ng/microliter) size (<200 nt)-enriched microRNA (miRNA) library were sequenced on an Ion Torrent Personal Genome Machine (PGM™) Sequencing platform at Life Sequencing S.L.
Bioinformatic processing of the Ion-Torrent miRNA reads
Using Prinseq , the Ion-Torrent miRNA reads were filtered out according to nucleotide length (min_length 15 nt; max_length 40 nt) and the presence of either the 30-mer IonA or the 23-mer P1 adapter sequences. Adapter sequences were removed using the fasxt_clipper tool from the Fastx-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit). The resulting reads were clustered using CD-HIT-454  setting an identity threshold of 0.98 and the accurate mode option g1. HIT-454 was designed to identify artificial duplicates from raw 454 sequencing reads, including exact duplicates and near identical duplicates. Script cdhit-cluster-consensus (v.13) of the CD-HIT suite of programs was run to derive consensus sequences for each of the miRNA clusters. This program is currently maintained by Dr. Li's group (http://weizhong-lab.ucsd.edu). The relative abundances of miRNAs with frequency count ≥ 100 were normalized by scaling the number of reads clustered by CD-HIT-454 to the total number of processed reads (220,548 for newborn + 389,064 for adult). To identify unique and shared sequences between the newborn and adult datasets, these newborn and adult miRNA clusters were compared between themselves using CD-HIT. Differential expression between miRNAs in these two datasets was assessed with NOISeq-sim  allowing a threshold of 0.9. miRNAs comprising ≥ 100 reads were subjected to BLAST analysis against miRBase (release 18, November 2011, which included 21643 mature miRNA products from 168 species) (http://www.mirbase.org) .
The search for miRNA targets
The precise rules and energetics for pairing between a miRNA and its mRNA target sites are not completely understood [104, 105]. A variety of computational algorithms aimed at predicting miRNA target genes incorporate rules for miRNA-mRNA interactions such as base pairing complementarity and favourable miRNA-mRNA duplex thermodynamics. Current prediction methods are diverse, both in approach and performance . We have employed the freely available target prediction, position-weighted local alignment miRanda algorithm (standalone version 3.3a) [80, 81] and the MapMi webserver (version 1.5.0-build 01, release March 2012) (http://www.ebi.ac.uk/enright-srv/MapMi)  to identify candidate miRNA target sites in 3'-UTR regions of 454 reads by base complementarity, and putative miRNA loci, respectively. MapMi is a tool designed to locate miRNA precursor sequences in existing genomic sequences, using potential mature miRNA sequences as input. miRanda is an algorithm for finding genomic targets for microRNAs that incorporates current biological knowledge on target rules and computes optimal sequence complementarity between a set of mature microRNAs and a given mRNA using a weighted dynamic programming algorithm. In addition, miRanda uses an estimate of the free energy of formation of the miRNA:mRNA duplex as a secondary filter . The following parameters were set to reduce the estimated false positives to ≤ 9% [80, 81]: total Score >100; ΔG of the intermediate duplex < −19 Kcal/mol; and output of 2 or more 454-contig targets.
The raw 454 GS FLX Titanium reads of C. s. simus adult and neonate venom gland transcriptomes, and the Ion Torrent PGM reads (miRNA sequences) of adult and neonate C. s. simus have been archived as Standard Flowgram Format (sff) files with the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra?term=SRA051956) under accession codes SRX143982 and SRX143985, SRX143983 and SRX143984, respectively.
Procedures for snake maintenance, sacrifice, and gland extraction in this study followed the Quality Management Protocols from the Instituto Clodomiro Picado, University of Costa Rica, and comply the Animal Welfare Law #7451, chapters II and III, and the Biodiversity Law, Decree 7788, Republic of Costa Rica. Research permission was allowed under Resolution ACG- SINAC- PI-012-2010 (Ministry of Environment of Costa Rica).
This work has been financed by grant BFU2010-17373 from the Ministerio de Economía y Competitividad, Madrid, Spain; PROMETEO/2010/005 from the Generalitat Valenciana; and projects 741-B2-093 from the Vicerrectoría de Investigación, Universidad de Costa Rica, CRUSA-CSIC (2009CR0021), and CYTED (206AC0281). We also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). JD is grateful to Eva Alloza (CIPF) and José Afonso Guerra-Assunçâo (EBI) for useful comments and advice regarding miRNA sequence analysis.
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