Desideri Fabio, D'Ambra Eleonora, Laneve Pietro, Ballarino Monica
Center for Life Nano- & Neuro-Science of Istituto Italiano di Tecnologia (IIT), Rome, Italy.
Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy.
Front Mol Biosci. 2022 Oct 19;9:1004746. doi: 10.3389/fmolb.2022.1004746. eCollection 2022.
Detecting RNA/RNA interactions in the context of a given cellular system is crucial to gain insights into the molecular mechanisms that stand beneath each specific RNA molecule. When it comes to non-protein coding RNA (ncRNAs), and especially to long noncoding RNAs (lncRNAs), the reliability of the RNA purification is dramatically dependent on their abundance. Exogenous methods, in which lncRNAs are transcribed and incubated with protein extracts or overexpressed by cell transfection, have been extensively used to overcome the problem of abundance. However, although useful to study the contribution of single RNA sub-modules to RNA/protein interactions, these exogenous practices might fail in revealing biologically meaningful contacts occurring and risk to generate non-physiological artifacts. Therefore, endogenous methods must be preferred, especially for the initial identification of partners specifically interacting with elected RNAs. Here, we apply an endogenous RNA pull-down to lncMN2-203, a neuron-specific lncRNA contributing to the robustness of motor neurons specification, through the interaction with miRNA-466i-5p. We show that both the yield of lncMN2-203 recovery and the specificity of its interaction with the miRNA dramatically increase in the presence of Dextran Sulfate Sodium (DSS) salt. This new set-up may represent a powerful means for improving the study of RNA-RNA interactions of biological significance, especially for those lncRNAs whose role as microRNA (miRNA) sponges or regulators of mRNA stability was demonstrated.
在特定细胞系统中检测RNA/RNA相互作用对于深入了解每个特定RNA分子背后的分子机制至关重要。对于非蛋白质编码RNA（ncRNAs），尤其是长链非编码RNA（lncRNAs），RNA纯化的可靠性极大地依赖于它们的丰度。外源性方法，即lncRNAs被转录并与蛋白质提取物一起孵育或通过细胞转染过表达，已被广泛用于克服丰度问题。然而，尽管这些外源性方法对于研究单个RNA亚模块对RNA/蛋白质相互作用的贡献很有用，但它们可能无法揭示发生的生物学上有意义的相互作用，并且有产生非生理性假象的风险。因此，内源性方法更受青睐，特别是对于最初鉴定与选定RNA特异性相互作用的伙伴。在这里，我们通过与miRNA-466i-5p相互作用，将内源性RNA下拉技术应用于lncMN2-203，这是一种对运动神经元特化的稳健性有贡献的神经元特异性lncRNA。我们表明，在存在硫酸葡聚糖钠（DSS）盐的情况下，lncMN2-203回收的产量及其与miRNA相互作用的特异性都显著增加。这种新方法可能是一种强大的手段，可用于改进对具有生物学意义的RNA-RNA相互作用的研究，特别是对于那些已被证明具有作为微小RNA（miRNA）海绵或mRNA稳定性调节剂作用的lncRNAs。
