Mapping effective microRNA pairing beyond the seed using abasic mutations

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base-pairing to complementary sites in mRNAs. The most important element for site recognition is the seed region (nucleotides 2-8 in the miRNA), but for a minority of sites pairing outside the seed increases efficiency, with the supplementary region (nucleotides 13-16) typically having the greatest impact. However, it is not clear how large an effect supplementary pairing has on target site efficiency, or which sites are most affected by it. Here, we use abasic modified nucleotides to disrupt pairing to residues 13 and 14 of miR-34a and measure the effect of this mutation compared to wild-type miR-34a on the cellular transcriptome using RNA-seq. We find that a subset of sites with predicted supplementary pairing are affected 24 hours after miRNA overexpression, with up to two-fold decreases in site repression. We show that miR-34a 3'-pairing is sensitive to GU wobble pairs in a position-specific manner and favors bulges in the miRNA over the target. These results were validated with luciferase reporter assays. Overall, this study demonstrates a novel methodological approach for elucidating the role of specific miRNA residues in target site selection, advancing our understanding of miRNA-mediated gene regulation. Overall design: To investigate the impact of supplementary pairing on microRNA targeting, we compared the effects on gene expression of wild-type miR-34a (miR-34a-WT) and a variant where miR-34a residues g13 and g14 were replaced by rSpacer abasic residues (miR-34a-RS). HEK293T cells were transfected with miR-34a-WT duplex, miR-34a-RS duplex or AllStars negative control (Qiagen). Total RNA was collected 24 h post-transfection and samples were analyzed for differential gene expression using bulk RNA-sequencing.