Characteristic analysis of a secondary structure of primary microRNA bound to a double-strand RNA-binding protein, NF90

Abstract

MicroRNAs (miRNA) are non-coding small RNAs that function as a suppressor for gene expression by binding to the 3’ untranslated region (3’UTR) of target messenger RNAs. We previously reported that Nuclear Factor 90 (NF90) and NF45 complex, a double-stranded RNA-binding protein, plays a negative regulator in miRNA biogenesis through a binding to primary miRNAs (pri-miRNAs) which are initially transcribed from miRNA gene. However, a common secondary structure of the NF90-targeted pri-miRNAs are still unclear. In this study, we attempted to characterize the structure of NF90 binding pri-miRNAs by informatics analysis and RNA-electrophoretic mobility shift assay (RNA-EMSA). We used genome-wide approaches including ENCODE and small RNA-seq to identify pri-miRNAs that are associated with NF90. As a result, we found that NF90-targerted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. To confirm this, we performed RNA-EMSA probed with mutated pri-miRNAs. Pri-miR-3173 and pri-miR-186, the NF90-targeted pri-miRNA, were mutated to unstable structure by making bulges in the stem structure, whereas pri-miR-200a, which exhibits low affinity to NF90, was changed to more stable structure by forming long duplex in the stem. Consequently, we found that the mutated pri-miR-3173 and pri-miR-186 lead to a reduction in binding activity of NF90 to the pri-miRNAs. On the other hand, the binding affinity of NF90 to pri-miR-200a was dramatically elevated by forming the long duplex in the structure. These results suggest that NF90 preferentially binds pri-miRNAs having less mismatches and highly stable stem structure.