Characteristic analysis of a secondary structure of primary microRNA bound to a double-strand RNA-binding protein, NF90
Characteristic analysis of a secondary structure of primary microRNA bound to a double-strand RNA-binding protein, NF90
复制标题
DOI：
10.2198/electroph.65.13
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Takuma Higuchi;R. Kiernan;S. Sakamoto
中科院分区：
文献类型：
--
作者：
Takuma Higuchi;R. Kiernan;S. Sakamoto
不清楚在本研究中，我们试图通过信息学分析和RNA电泳迁移率变动分析（RNA-EMSA）来表征NF 90结合pri-miRNAs的结构。我们使用全基因组方法，包括ENCODE和small RNA-seq来鉴定与NF 90相关的pri-miRNAs。因此，我们发现NF 90靶向的pri-miRNAs是高度稳定的，与所有pri-miRNAs相比具有更低的自由能和更少的错配。为了证实这一点，我们用突变的pri-miRNAs进行了RNA-EMSA探测。靶向NF 90的pri-miRNA pri-miR-3173和pri-miR-186通过在茎结构中形成凸起而突变为不稳定结构，而对NF 90表现出低亲和力的pri-miR-200 a通过在茎中形成长双链体而改变为更稳定的结构。因此，我们发现突变的pri-miR-3173和pri-miR-186导致NF 90与pri-miRNA的结合活性降低。另一方面，NF 90与pri-miR-200 a的结合亲和力通过在结构中形成长双链体而显著提高。这些结果表明NF 90优先结合具有较少错配和高度稳定茎结构的pri-miRNA。
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.