MicroRNA-mediated network redundancy is constrained by purifying selection and contributes to expression robustness

MicroRNAs (miRNAs) act as trans-acting elements and form composite feedforward circuits (cFFCs) in conjunction with transcription factors (TFs), leading to redundancy in network motifs and influencing gene expression regulation. However, the maintenance of miRNA-mediated regulatory redundancy and its impact on gene expression evolution remain elusive. Using public ChIP-seq/ChIP-chip and ATAC-seq data, we constructed miRNA-mediated cFFCs in Drosophila melanogaster embryos and found that most of predicted miRNA targets are involved in redundant cFFCs. Population genetics analysis revealed that highly redundant cFFCs are subjected to stronger purifying selection than low-redundant or non-redundant cFFCs. This redundancy negatively correlates with expression divergence between D. melanogaster and D. simulans , and is associate with expression stability in D. melanogaster embryos under heat shock. Moreover, highly redundant cFFCs mainly target older genes or young genes with broader expression. Our findings emphasize the biological significance of miRNA-mediated cFFC redundancy in mediating the robustness of gene expression during evolution.