Acts upstream of or within, negative effect
The acts upstream of or within, negative effect annotation relation is used when the mechanism relating the gene product's activity to the Biological Process is not known. However, the observed experimental outcome indicates that, ultimately, the gene product's activity has a negative effect on the process. Annotations using this relation often come from mutant phenotypes for which further characterization has not been performed, but the authors wish to report the effect that the gene has on a process.
Examples of Usage
- The zebrafish miR-462/miR-731 cluster is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations.
- Overexpression of miR-462 and miR-731 represses cell proliferation through blocking cell cycle progress of DNA replication, and induces apoptosis.
- Summary of the paper:
- The transcription factor HIF1-alpha is hydroxylated at proline residues under normal oxygen conditions ("normoxia"), which targets it for proteosomal degradation. Hence under normal conditions its half life is very short.
- Under limiting O2 conditions, the prolyl hydroxylase activity is reduced, and HIF1-alpha is stabilized, and interacts with HIF1-beta.
- The HIF1-alpha/beta dimer activates hundreds of genes involved in hypoxia adaptation via their DNA binding transcription activator activity, binding to HRE (hypoxia-responsive elements)
- Hundreds of genes are also down-regulated in a HIF1-dependent manner, but without direct HIF1-promoter binding
- Deep sequencing of small RNA libraries identified 129 and and 212 novel miRNAs under normoxia and hypoxia conditions, 102 of which in common to both sets (NOTE: these miRNAs were not further studied).
- To identify known miRNA, the authors looked into the miRBase, that contained 247 known miRNAs. Seven differentially regulated miRNAs were identified: 5 upregulated and 2 downregulated (Table 3).
- dre-miR-462 and dre-miR-731 are both on a cluster on Chr8 and were shown to be co-upregulated in response to hypoxia (Fig 1) and upon overexpression of HIF1 (Fig 2A), and decreased by knockdown of HIF1
- A HIF1 binding site was identified 22 nt upstream from the Chr 8 cluster
- Targets: 2050 putative targets of the 8 miRNAs that belong to the same Chr 8 cluster were predicted - involved in multiple pathways and processes. Two of these targets were selected: ddx5 (miR-462 target) and ppmda (miR-731 target)
- Luciferase assay with the 3'UTR sequence of the ddx5 and ppmda mRNAs showed that these 3'UTR are sufficient to decrease expression of luciferase (Fig 3)
- ddx5 and mRNA and protein were downregulated by miR-462 mimics and upregulated by miR-462 inhibitors, indicating that the miRNA does contribute to the regulation of expression level of ddx5
- Assayed effect of miR-462 and miR-731 on proliferation and apoptosis.
- Both miRNAs significantly repressed proliferation (Fig 4A), blocking cell cycle progression at S phase (Fig 4B,C)
- Overexpression of miR-462 and miR-731 increased apoptosis (Fig 4E) - in a caspase-3 independent manner
- miR-462 'acts upstream of or within, negative effect' cell proliferation
Quality Control Checks
Last reviewed: March 9, 2018