Background: The discovery that a plant microRNA (miRNAs) from rice (Oryza sativa
miR168a) can modify post-transcriptional expression of the mammalian. Low-Density Lipoprotein
Receptor Adaptor Protein 1 (LDLRAP1) gene highlights the potential for cross-kingdom miRNAmRNA
Objective: To investigate whether common variants of the conserved miR168a family have the capability
for similar cross-kingdom regulatory functions, we selected sequences from three dietary plant
sources: rice (Oryza sativa), tomato (Solanum lycopersicum), apple (Malus domestica) and compared
their ability to regulate human LDLRAP1 expression.
Methods: Target prediction software intaRNA and RNAhybrid were used to analyze and calculate
the energy and alignment score between the miR168a variants and human LDLRAP1 mRNA. An in
vitro cell-based Dual-Luciferase® Reporter Assay (pmirGLO, Promega), was then used to validate
the miRNA-mRNA interaction experimentally.
Results: Computational analyses revealed that a single nucleotide difference at position 14 (from the
5’ end of the miRNA) creates a G:U wobble in the miRNA-mRNA duplex formed by tomato and apple
miR168a variants. This G:U wobble had only a small effect on the free energy score (-33.8–34.7
kcal/mol). However, despite reasonable hybridization energy scores (<-20 kcal/mol) for all miR168a
variants, only the rice miR168a variant lacking a G:U wobble significantly reduced LDLRAP1 transcript
expression by 25.8 + 7.3% (p<0.05), as measured by relative luciferase activity.
Conclusion: In summary, single nucleotide differences at key positions can have a marked influence
on regulatory function despite similar predicted energy scores and miRNA-mRNA duplex structures.