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Common Variants of the Plant microRNA-168a Exhibit Differing Silencing Efficacy for Human Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1)

Author(s):

Claudia Lang, Sakuntala Karunairetnam, Kim R. Lo, Andrew V. Kralicek, Ross N. Crowhurst, Andrew P. Gleave, Robin M. MacDiarmid and John Ronald Ingram*   Pages 1 - 5 ( 5 )

Abstract:


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 interactions. 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.

Keywords:

miRNA, LDLRAP1, cross-kingdom regulation, miR-168a, gene-reporter assay, miRNA variants

Affiliation:

The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142, The New Zealand Institute for Plant & Food Research Ltd, Auckland 1142



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