Competing targets of microRNA-608 affect anxiety and hypertension.
Bottom Line: We found that changing a single miRNA-target interaction can simultaneously affect multiple other miRNA-target interactions and modify physiological phenotype.Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension.We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.
Affiliation: The Silberman Institute of Life Sciences and The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, The Edmond Safra Campus, Givat Ram, Jerusalem 91904, Israel.Show MeSH
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Mentions: Predictably, miR-608 shows thousands of potential targets (miRNAwalk: http://www.umm.uni-heidelberg.de/apps/zmf/miRNAwalk). Of those, the validated miR-608 targets Rho GTPase CDC42 (19) and IL6 (20) are predictably involved in anxiety and parasympathetic signaling. Bioinformatics analysis (RNAhybrid, http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) predicted relatively tight binding to miR-608 for the C-allele and the A-allele sequences (−31.4 and −25.8 Kcal/mol), CDC42 (−26.4 Kcal/mol) and miR-132–AChE interaction (−17.3 Kcal/mol) (Fig. 2A and B). To experimentally measure miR-608–target association, we adapted an in vitro surface plasmon resonance (SPR) assay (22) for hybridization tests. Given that miRNA–target interactions may involve longer regions than the seed itself (1), we immobilized biotin-linked 30-mer RNA sequences of the corresponding regions in the major C-allele of AChE or CDC42 to SPR chips and injected a 25-mer RNA oligonucleotide with the miR-608 sequence. This demonstrated a ∼15-fold reduction in the affinity of miR-608 to the minor A-allele compared with the C-allele AChE sequences (KD of 50.9 versus 3.1 nm, Fig. 2C and D and Supplementary Material, Fig. S1H), indicating weakened A-allele AChE–miR-608 interaction. CDC42-miR-608 and AChE–miR-132 presented intermediate affinities (15.8 and 18.8 nm, Fig. 2E and F), predicting a hierarchical binding preference of miR-608 to the C-allele AChE, CDC42 and the A-allele AChE target sites (Fig. 2G and H).Figure 2.
Affiliation: The Silberman Institute of Life Sciences and The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, The Edmond Safra Campus, Givat Ram, Jerusalem 91904, Israel.