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Competing targets of microRNA-608 affect anxiety and hypertension.

Hanin G, Shenhar-Tsarfaty S, Yayon N, Yau YH, Hoe YY, Bennett ER, Sklan EH, Rao DC, Rankinen T, Bouchard C, Geifman-Shochat S, Shifman S, Greenberg DS, Soreq H - Hum. Mol. Genet. (2014)

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.

View Article: PubMed Central - PubMed

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.

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Quantified miR-608–target interactions. (A) Target and miRNA RNA oligonucleotides sequences. Seed regions are colored. (B) Predicted structures and binding energy of miR-608 with AChE's C-allele and A-allele and CDC42, and of miR-132 with AChE. (C–E) SPR sensograms showing binding of miR-608 to the C-allele and A-allele of AChE and CDC42 targets. Biotinylated target RNA oligonucleotides were immobilized to a streptavidin chip, and increasing concentrations (0.3125, 0.625, 1.25, 2.5, 5 and 10 μm) of miRNA oligonucleotides were injected over the chip. (F) SPR sensorgrams showing miR-132–AChE binding. (G) SPR dissociation slopes of the indicated interactions. (H) ka and kd values for the SPR reactions.
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DDU170F2: Quantified miR-608–target interactions. (A) Target and miRNA RNA oligonucleotides sequences. Seed regions are colored. (B) Predicted structures and binding energy of miR-608 with AChE's C-allele and A-allele and CDC42, and of miR-132 with AChE. (C–E) SPR sensograms showing binding of miR-608 to the C-allele and A-allele of AChE and CDC42 targets. Biotinylated target RNA oligonucleotides were immobilized to a streptavidin chip, and increasing concentrations (0.3125, 0.625, 1.25, 2.5, 5 and 10 μm) of miRNA oligonucleotides were injected over the chip. (F) SPR sensorgrams showing miR-132–AChE binding. (G) SPR dissociation slopes of the indicated interactions. (H) ka and kd values for the SPR reactions.

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.


Competing targets of microRNA-608 affect anxiety and hypertension.

Hanin G, Shenhar-Tsarfaty S, Yayon N, Yau YH, Hoe YY, Bennett ER, Sklan EH, Rao DC, Rankinen T, Bouchard C, Geifman-Shochat S, Shifman S, Greenberg DS, Soreq H - Hum. Mol. Genet. (2014)

Quantified miR-608–target interactions. (A) Target and miRNA RNA oligonucleotides sequences. Seed regions are colored. (B) Predicted structures and binding energy of miR-608 with AChE's C-allele and A-allele and CDC42, and of miR-132 with AChE. (C–E) SPR sensograms showing binding of miR-608 to the C-allele and A-allele of AChE and CDC42 targets. Biotinylated target RNA oligonucleotides were immobilized to a streptavidin chip, and increasing concentrations (0.3125, 0.625, 1.25, 2.5, 5 and 10 μm) of miRNA oligonucleotides were injected over the chip. (F) SPR sensorgrams showing miR-132–AChE binding. (G) SPR dissociation slopes of the indicated interactions. (H) ka and kd values for the SPR reactions.
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Related In: Results  -  Collection

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DDU170F2: Quantified miR-608–target interactions. (A) Target and miRNA RNA oligonucleotides sequences. Seed regions are colored. (B) Predicted structures and binding energy of miR-608 with AChE's C-allele and A-allele and CDC42, and of miR-132 with AChE. (C–E) SPR sensograms showing binding of miR-608 to the C-allele and A-allele of AChE and CDC42 targets. Biotinylated target RNA oligonucleotides were immobilized to a streptavidin chip, and increasing concentrations (0.3125, 0.625, 1.25, 2.5, 5 and 10 μm) of miRNA oligonucleotides were injected over the chip. (F) SPR sensorgrams showing miR-132–AChE binding. (G) SPR dissociation slopes of the indicated interactions. (H) ka and kd values for the SPR reactions.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus