Unusual stability of messenger RNA in snake venom reveals gene expression dynamics of venom replenishment

Abstract

Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3-7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies.

Figure 1. PCR amplification of cDNA constructed from venom gland and venom mRNA.

Qualitatively similar PCR products were amplified from cDNA from venom gland (A) or venom (B), using primers complementary to Bitis arietans venom metalloproteinases (SVMP), phospholipase A₂ (PLA₂), serine protease (SP), C-type lectins (CTL), vascular endothelial growth factor (VEGF), L-amino acid oxidase (LAO), Kunitz inhibitors (KTI), protein disulphide isomerase (PDI) and QKW inhibitory peptides (QKW). Molecular weight markers (M) are shown to the left.

Figure 2. Venom mRNA expression profiles during venom re-synthesis.

Relative expression profiles of six venom transcripts (snake venom metalloproteinase, serine protease, C-type lectin, Kunitz inhibitors, protein disulphide isomerase and QKW inhibitory peptide) across the time course of venom re-synthesis were determined by relative quantitative PCR (qPCR). The average fold changes in % venom protein gene expression for A) 3 Ghanaian B. arietans specimens and B) 5 Nigerian B. arietans specimens was normalised against three references genes (β actin, glyceraldehyde-3-phosphate dehydrogenase and heat shock protein) and indicate that the expression of venom protein genes peaks on day 0–3 to 0–7 (Red = day 0–1, orange = day 0–3, blue = day 0–7, green = mature venom).

Figure 3. HPLC and mass spectrometry analysis of pooled venom samples from Ghana and Nigeria during venom re-synthesis.

HPLC-MS/MS identification of proteins from pooled Ghanaian and Nigerian venom samples showed that very little quantitative changes in the protein composition of venom during protein re-synthesis. We identified by mass spectrometry a range of proteins including disintegrins (DISI, peaks 3, 4), Kunitz inhibitors (KI, peak 8), PLA₂ (peak 13), cysteine-rich secretory proteins (Cyst, peak 14), serine proteases (SerProt, peaks 15–18), CTLs (peaks 22, 25) and PI (peak 26, 27) and PIII SVMPs (peak 29) which were present in all venom samples from day 0–1 to mature venom.

Figure 4. Individual venom protein profiles during venom re-synthesis by HPLC and 1D SDS-PAGE.

Analysis of venom samples extracted on day 0–1, day 0–3, day 0–7 and mature venom for each individual specimen across the time course of venom re-synthesis by HPLC showed very little quantitative differences in protein profile. 1D-SDS-PAGE panels are shown to the right of HPLC profiles which confirm observations by HPLC. Molecular weight markers (M) are shown to the far right.

Figure 5. Correlation between natural venom protein concentration and venom yield.

The natural protein concentration of venoms and venom yield of venoms extracted from day 0–1, 0–3, 0–7 were analysed. An inverse relationship between the protein concentration of venom (A) and the % maximum venom yield (B) over time was observed, indicating that venom protein concentration peaks between day 0–3 and day 0–7.

Figure 6. Venom enzyme activity profiles during venom re-synthesis.

Gelatin zymography of venom samples extracted at day 0–1, 0–3, 0–7 and mature venom and reconstituted at natural protein concentration was used to assess enzyme activity of venom (A). The enzymatic degradation of substrate by venom extracted on day 0–1 was equal to mature venom. Panel B shows a range-finding zymogram which shows that the dynamic range of this assay (arrow above indicate the range of venom quantity used in panel A). Molecular weight markers (M) are shown to the left.

Figure 7. Prolonged stability of mRNA in lyophilised venom.

PCR amplification of a range of venom protein transcripts including snake venom metalloproteinases (SVMP), phospholipase A₂ (PLA₂), serine protease (SP), C-type lectins (CTL), vascular endothelial growth factor (VEGF), L-amino acid oxidase (LAO), Kunitz inhibitors (KTI), protein disulphide isomerase (PDI) and QKW inhibitory peptides (QKW) from mRNA isolated from a venom sample extracted and lyophilised in 1984.