Cis sequences regulate the expression of the primate-specific hsa-miR-608

‘CholinomiR’ microRNA controllers of acetylcholine signaling regulate stress responses and inflammation, but the underlying molecular mechanisms and their impact on the brain’s stress reactions are incompletely understood. Notably, the primate-specific ‘CholinomiR’ hsa-miR-608 (miR-608) derives from an intron of the inflammation-associated SEMA4G gene and targets acetylcholinesterase (AChE). A single nucleotide polymorphism (SNP) in the 3’-untranslated region of AChE mRNA weakens AChE/miR-608 interaction; human carriers of the rare allele of this SNP show elevated anxiety, inflammation and blood pressure (Hanin 2014) but not stress symptoms (Lin et al., 2016). To map regulatory regions within miR-608’s native location, we expressed miR-608 and its flanking sequences in human and murine cell lines. This identified a 150 base pairs long sequence 5` to the miR-608 stem-loop which up-regulated its levels 100-fold, contains a TATA box and enables miR-608 expression independently of its host gene. Moreover, we identified sequences 3` to the miR-608 stem-loop which decline its expression. We further engineered mice carrying miR-608 in the 3rd intron of the murine SEMA4G gene, with 250 nucleotides-long flanking regions around the stem-loop. These mice expressed miR-608 in similar tissues and levels compared to humans. Intriguingly, brain multi-electrode array recordings of these engineered mice demonstrated female-accentuated medial prefrontal cortex activity in response to cholinergic stimulation, in agreement with recently established sex-related differences in cholinergic signaling in human brains from mental disease patients (Lobentanzer et al., 2019). We conclude that sequences flanking miR-608 can regulate its sex-related expression in rodents and primates, indicating their functionality.