Structural and Functional Analyses of Hub Micro RNAs in An Integrated Gene Regulatory Network of Arabidopsis

MicroRNAs(miRNAs)are trans-acting small regulatory RNAs that work coordinately with transcription factors(TFs)to shape the repertoire of cellular m RNAs available for translation.Despite our growing knowledge of individual plant miRNAs,their global roles in gene regulatory networks remain mostly unassessed.Based on interactions obtained from public databases and curated from the literature,we reconstructed an integrated miRNA network in Arabidopsis that includes 66 core TFs,318 miRNAs,and 1712 downstream genes.We found that miRNAs occupy distinct niches and enrich miRNA-containing feed-forward loops(FFLs),particularly those with miRNAs as intermediate nodes.Further analyses revealed that miRNA-containing FFLs coordinate TFs located in different hierarchical layers and that intertwined miRNA-containing FFLs are associated with party and date miRNA hubs.Using the date hub MIR858A as an example,we performed detailed molecular and genetic analyses of three interconnected miRNA-containing FFLs.These analyses revealed individual functions of the selected miRNA-containing FFLs and elucidated how the date hub miRNA fulfills multiple regulatory roles.Collectively,our findings highlight the prevalence and importance of miRNA-containing FFLs,and provide new insights into the design principles and control logics of miRNA regulatory networks governing gene expression programs in plants.