Sweetapple Lara, Kosek David M, Banijamali Elnaz, Becker Walter, Müller Juliane, Karadiakos Christina, Baronti Lorenzo, Guzzetti Ileana, Schritt Dimitri, Chen Alan, Andersson Emma R, Petzold Katja
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177, Sweden.
Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, 171 77 Sweden.
Nucleic Acids Res. 2025 Jul 8;53(13). doi: 10.1093/nar/gkaf633.
MicroRNAs (miRs) regulate gene expression post-transcriptionally by guiding Argonaute (AGO) proteins to target mRNAs. Efficiently predicting the repressive effects of miRNAs remains limited largely due to an incomplete understanding of how mRNA:miR structure affects function. Using EMSAs, structural probing, luciferase reporter assays, and transcriptome analysis, we investigated the structural, biophysical, and functional interaction between the human tumour suppressor miR-34a and 12 mRNA targets. Comparison of isolated mRNA:miRNA duplexes and those bound within functional AGO2 revealed that while the binary duplex largely predicts AGO2-associated affinity and structure, AGO2 bidirectionally modulates binding by attenuating strong interactions and stabilising weaker ones. Furthermore, we show that the impact of supplementary pairing is more pronounced in targets with shorter seeds compared to those with full-length seeds and confirm this effect in a transcriptome-wide analysis. Finally, we identified three structural groups of mRNA:miR-34a-AGO2 complexes, adopting either a symmetrical structure, or a bulge on the mRNA or miR side. miR-bulged complex repression was strongly linked to mRNA:miR affinity, whereas mRNA-bulged complexes showed no such correlation. Our results thus identify structural and biophysical characteristics of mRNA:miR duplexes that contribute to repression efficacy, revealing a hierarchy of seed type, structure, and affinity that determine repression efficiency.
微小RNA（miR）通过引导AGO蛋白靶向mRNA在转录后水平调控基因表达。由于对mRNA:miR结构如何影响功能的理解不完整，有效预测miRNA的抑制作用仍然受到很大限制。我们使用电泳迁移率变动分析（EMSA）、结构探测、荧光素酶报告基因检测和转录组分析，研究了人类肿瘤抑制因子miR-34a与12个mRNA靶标的结构、生物物理和功能相互作用。对分离的mRNA:miRNA双链体和功能性AGO2中结合的双链体进行比较，结果表明，虽然二元双链体在很大程度上可预测AGO2相关的亲和力和结构，但AGO2通过减弱强相互作用并稳定弱相互作用来双向调节结合。此外，我们发现与具有全长种子的靶标相比，补充配对对具有较短种子的靶标的影响更为明显，并在全转录组分析中证实了这一效应。最后，我们确定了mRNA:miR-34a-AGO2复合物的三个结构组，它们分别具有对称结构，或在mRNA侧或miR侧有一个凸起。miR凸起复合物的抑制作用与mRNA:miR亲和力密切相关，而mRNA凸起复合物则没有这种相关性。因此，我们的研究结果确定了有助于抑制效果的mRNA:miR双链体的结构和生物物理特征，揭示了决定抑制效率的种子类型、结构和亲和力的层次结构。
