Altered RNome expression in Murine Gastrocnemius Muscle following Exposure to Jararhagin, a Metalloproteinase from Bothrops jararaca Venom

Abstract

Small RNAs (sRNAs) and microRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs that regulate gene expression in eukaryotes. Experiments in mice and humans have revealed that a typical small RNA can affect the expression of a wide range of genes, implying that small RNAs function as global regulators. Here, we used small RNA deep sequencing to investigate how jararhagin, a metalloproteinase toxin produced from the venom of Bothrops jararaca, affected mmu-miRNAs expression in mice 2 hours (Jar 2hrs) and 24 hours (Jar 24hrs) after injection compared to PBS control. The findings revealed that seven mmu-miRNAs were substantially differentially expressed (p value (p (Corr) cut-off 0.05, fold change ≥ 2) at 2 hrs after jararhagin exposure and that the majority of them were upregulated when compared to PBS. In contrast to these findings, a comparison of Jar 24hrs vs. PBS 24hrs demonstrated that the majority of identified mmu-miRNAs were downregulated. Furthermore, the studies demonstrated that mmu-miRNAs can target the expression of several genes involved in the MAPK signaling pathway. The steady antithetical regulation of mmu-miRNAs may correlate with the expression of genes that trigger apoptosis via MAPK in the early stages, and this effect intensifies with time. The findings expand our understanding of the effects of jararhagin on local tissue lesions at the molecular level.

Keywords: jararhagin; small RNAs; venom.

Figure 1

Venn diagram of known sRNA expression among groups.

Figure 2

Differences in known small RNA (sRNA, n = 22) expression levels from gastrocnemius muscle samples obtained after 2 hrs and 24 hrs of challenge with jararhagin and PBS. The sample-clustering tree is displayed to the left and the sRNA clustering tree is above forming 3 clusters as indicated by black, purple, and yellow colors. The color scale at the top indicates the relative expression levels of sRNA across all samples. Red indicates a high expression level and blue indicates a low expression level. Each column represents one known sRNA, and each row represents one sample.

Figure 3

Principal component analysis (PCA) plot showing distances among the four groups based on the profile of the 22 significantly expressed known small RNAs. The shaded circles highlighted (yellow, blue, gray, red) refer to the clusters formed from small RNA molecular data.

Figure 4

Differences in novel small RNA (sRNA, n = 17) expression levels from gastrocnemius muscle samples obtained after 2 and 24 hrs of challenge with jararhagin and PBS. The sample-clustering tree is displayed to the left, and the sRNA clustering tree is above forming two clusters as indicated by purple and yellow colors. The color scale at the top indicates the relative expression levels of sRNA across all samples Red indicates a high expression level and blue indicates a low expression level. Each column represents one known sRNA and each row represents one sample.

Figure 5

Differences in mature mmu-miRNA (n = 7) expression levels from gastrocnemius muscle samples obtained after 2 and 24 hrs of challenge with jararhagin and PBS. The sample clustering tree is displayed to the left and the sRNA clustering tree is above. The color scale at the top indicates the relative expression levels of sRNA across all samples. Red indicates a high expression level and blue indicates a low expression level. Each column represents one known sRNA, and each row represents one sample.

Figure 6

Principal component analysis (PCA) plot showing distances among the four groups based on the profile of the seven significantly expressed mmu-miRNAs.

Figure 7

KEGG pathway for MAPK signaling pathway. The output of miRWalk analysis demonstrating the MAPK signaling pathway significantly enriched for significantly dysregulated mature mmu-miRNAs. Red boxes shaded in green denote predicted target genes. Gray shades indicate scaffolding.