miR858a-encoded peptide, miPEP858a, interacts with the miR858a promoter and requires the C-terminus for associated...

MicroRNAs (miRNAs) are key regulators of gene expression and typically processed from primary transcripts (pri-miRNAs). Recent discoveries highlight that certain pri-miRNAs also encode miRNA-encoded peptides (miPEPs), which influence miRNA function. However, the molecular mechanisms underlying miPEP activity, including the specific domains or essential amino acid residues required for their function, remain largely unexplored. In this study, we elucidated that the pri-miR858a-derived peptide, miPEP858a, directly interacts with the promoter of the MIR858 gene in Arabidopsis (Arabidopsis thaliana). Notably, the C-terminal region of miPEP858a, composed of 14 amino acid residues, is critical for its functionality. Through DNA-protein interaction assays, including yeast 1-hybrid, chromatin immunoprecipitation (ChIP-qPCR), electrophoretic mobility shift assay, and promoter-reporter analyses, we demonstrated that miPEP858a binds to a specific region within the MIR858 promoter. Exogenous application of a synthetic peptide corresponding to the C-terminal region of miPEP858a resulted in enhanced MIR858 expression, leading to phenotypic changes similar to those observed with the full-length miPEP858a. Moreover, the truncated C-terminal peptide was able to complement mutant plants lacking endogenous miPEP858a, emphasizing its role in regulating miR858a expression and downstream target genes involved in flavonoid biosynthesis and plant development. These findings suggest that the full-length miPEP858a may not be necessary for its biological function, with the C-terminal region being sufficient to modulate miRNA expression. This discovery reveals opportunities for identifying functional domains in other miPEPs, potentially reducing peptide synthesis costs, and offering a more efficient strategy for enhancing agronomic traits in crop plants without the need for complex biotechnological interventions.