Method and validation of stoichiometric reversal in miR-140 miRNAs. (A) Schematic of miR-140-3p and -5p miRNA asymmetry by Argonaute (Ago) loading. The schematic shows that the three miR-140 miRNAs, miR-140-5p, miR-140-3p.1, and miR-140-3p.2, are derived from Drosha cleavage of pri-miR-140 and subsequent cleavage of the loop region of the pre-miR-140 by Dicer. Note that two miR-140-3p miRNAs are produced (miR-140-3p.1 and -3p.2) from the -3p arm because Dicer cleavage occurs at two sites. The three miRNAs are asymmetrically loaded to Ago mostly dependent on the nature of the 5′ ends. Due to the preferential loading of miRNA strands with the 5′ nucleotides U or A, miR-140-3p miRNAs are more efficiently loaded to Ago than miR-140-5p that starts with C, a nonpreferred nucleotide for Ago loading. (B) Strategy to change the stoichiometry of miR-140-3p and -5p miRNAs. Blue bases indicate the mature miRNA sequences. Arrows indicate the first nucleotides of mature miRNAs. Base pairs in orange indicate where the mutations were made in each mouse model. The first model was created by altering the 5′ nt of the miR-140-3p strand to C and G from U and A, respectively, which are nonpreferential for Ago-loading; this model was designated as miR-140-CG. The 3′ end of miR-140-5p was also altered to maintain the hairpin loop structure of the pri-miR-140 allowing for normal processing by Drosha and Dicer and to provide thermodynamic stability of the 5′ end of miR-140-3p of the miR-140-5p/-3p duplex upon cleavage. In the second model, the mutation from C to U of the 5′ nt of miR-140-5p was designed to increase preferential Ago-loading of miR-140-5p; this model was designated as miR-140-C > T. (C) Quantification of mature miR-140-3p, miR-140-5p, let-7a-5p, and pri-miR-140 in primary rib chondrocytes of homozygous miR-140-CG (CG), miR-140-C > T (C > T), and their littermate wild-type control (WT). Pri-miR-140 was normalized by Actb. miRNAs were normalized by U6. miR-140-CG and miR-140-C > T mice show significantly increased miR-140-5p and significantly diminished miR-140-3p expression, whereas the level of pri-miR-140 or let-7a-5p (a control miRNA) was not significantly altered. Mean ± SEM with individual data is shown. n = 5–8 from three biological replicates, Student's t-test versus WT. (D) Quantification of Ago2-associated miR-140 miRNAs in homozygous miR-140-CG and wild-type littermate control (WT). The relative miR-140-3p/-5p ratio both in total RNA preparations (Input) and RNA-immunoprecipitated fraction with Ago2 antibody was reversed in CG chondrocytes. The P-values indicate the significance from comparing WT and CG. n = 6, Student's t-test.
