Atropa belladonna Expresses a microRNA (aba-miRNA-9497) Highly Homologous to Homo sapiens miRNA-378 (hsa-miRNA-378); both miRNAs target the 3'-Untranslated Region (3'-UTR) of the mRNA Encoding the Neurologically Relevant, Zinc-Finger Transcription Factor ZNF-691

Abstract

Recent advances in ethnobotanical and neurological research indicate that ingested plants from our diet may not only be a source of nutrition but also a source of biologically relevant nucleic-acid-encoded genetic information. A major source of RNA-encoded information from plants has been shown to be derived from small non-coding RNAs (sncRNAs) such as microRNAs (miRNAs) that can transfer information horizontally between plants and humans. In human hosts, the 3'-untranslated region (3'-UTR) of messenger RNAs (mRNAs) is targeted by these miRNAs to effectively down-regulate expression of that mRNA target in the host CNS. In this paper, we provide evidence that the Atropa belladonna aba-miRNA-9497 (miRBase conserved ID: bdi-miRNA-9497) is highly homologous to the CNS-abundant Homo sapiens miRNA-378 (hsa-miRNA-378) and both target the zinc-finger transcription factor ZNF-691 mRNA 3'-UTR to down-regulate ZNF-691 mRNA abundance. We speculate that the potent neurotoxic actions of the multiple tropane alkaloids of Atropa belladonna may be supplemented by the neuroregulatory actions of aba-miRNA-9497 on ZNF-691, and this may be followed by the modulation in the expression of ZNF-691-sensitive genes. This is the first example of a human brain-enriched transcription factor, ZNF-691, targeted and down-regulated by a naturally occurring plant microRNA, with potential to modulate gene expression in the human CNS and thus contribute to the neurotoxicological-and-psychoactive properties of the Atropa belladonna species of the deadly nightshade Solanaceae family.

Keywords: Atropa belladonna; Atropine; Trans-kingdom miRNA signaling; Zinc-finger protein ZNF-691; aba-miRNA-9497; hsa-miRNA-378.

Fig. 1

The flowers, berries, foliage, and roots of Atropa belladonna, a perennial herbaceous plant of the deadly nightshade family Solanaceae, contain: (i) a series of tropane alkaloids including atropine, scopolamine (hyoscine), and hyoscyamine that are highly neurotoxic, neuroactive, and psychoactive; and (ii) a complex array of Atropa belladonna-enriched microRNA (miRNA) species some of which show structural and functional homology to human brain and retinal miRNAs. Photo courtesy of Adobe Stock; https://gardenofgodsandmonsters.wordpress.com/2013/07/17/the-inflexible-unturnable-one-atropa-bella-donna (Simms https://gardenofgodsandmonsters.wordpress.com/2013/07/17/the-inflexible-unturn able-one-atropa-belladonna/; Kwakye et al. 2018)

Fig. 2

The mature aba-miRNA-9497 and hsa-miRNA-378 species; homologous structure and function; (a) sequence of the 111-nt aba-miRNA-9497 precursor and the mature 21 nucleotide (nt) aba-miRNA-9497 underlined in black and overlaid in yellow; (b) schematic depiction of extensive hydrogen bonding between the 21-nt aba-miRNA-9497 and the 21-nt human brain hsa-miRNA-378 indicates > 76% homology of hsa-miRNA-378 to aba-miRNA-9497; the energy of association (E_A) for either hsa-miRNA-378 to aba-miRNA-9497 to its ZNF-691 mRNA 3′-UTR target is predicted to be about − 21.5 kcal/mol (http://www.mirbase.org/; ver 22.0; 14 May 2019); 21-nt microRNAs of either plants or animals are the most evolutionarily ancient class of single-stranded non-coding RNA (sncRNA) species and among the most conservative ribonucleic acid sequences known (Ambros ; Li et al. ; Zhao et al. 2018); (c, d) hsa-miRNA-378 or aba-miRNA-9497-ZNF-691 mRNA 3′-UTR functionality assay: in these experiments, the entire 678-nt ZNF-691 3′-UTR ligated into the Nhe1-Xho1 restriction site of the pLightSwitch-Luciferase Reporter vector and along with sequence-scrambled (sc) controls was transfected into human neuronal-glial (HNG) cells in primary culture using TransFectin™ cationic lipid reagent (Cat no. 1703352) according to the manufacturer’s protocol (Biorad Life Science Research, Hercules CA, USA; Zhao et al. 2019); TargetScan ver 7.2 was used to predict miRNA-recognition features (highlighted in yellow; seed sequence underlined); (e) as indicated HNG cells (2 weeks in culture; 60–70% confluent; containing the pLightSwitch-Luciferase Reporter vector and scrambled controls) were next incubated with hsa-miRNA-378, a hsa-miRNA-378sc (containing a control scrambled hsa-miRNA-378 sequence), aba-miRNA-9497, and a aba-miRNA-9497sc (containing a control scrambled aba-miRNA-9497 sequence); the strength of the induced luciferase signal (relative luciferase yield) indicated a strong and selective interaction of hsa-miRNA-378 or aba-miRNA-9497 with the ZNF-691 3′-UTR; either aba-miRNA-5303 or aba-miRNA-7997 (Supplementary file S1) or at least 6 other plant or animal miRNAs selected at random showed no such specific interaction with the ZNF-691-3′-UTR (data not shown); the results are presented in bar graph format in panel (f); the results suggest that both hsa-miRNA-378 or aba-miRNA-9497 (but not their scrambled control sequences) strongly interact with the ZNF-691 3′-UTR to significantly down-regulate ZNF-691 expression to less than 0.18-fold of homeostatic control values; consequently hsa-miRNA-378 or aba-miRNA-9497 might be expected to modulate the down-regulation of ZNF-691-regulated gene expression (Guo et al. ; Jaber et al. ; Zhao et al. 2018); a dashed horizontal line at 1.0 is included for clarity and comparison and indicates the effect of control miRNAs on relative luciferase yield; N = 3 to 5 experiments for each miRNA; *p < 0.001 (ANOVA)