Faculté des Sciences Médicales et Paramédicales, Aix Marseille Univ, CNRS, INP.
Faculté des Sciences Médicales et Paramédicales, Aix Marseille Univ, CNRS, INP;
J Vis Exp. 2021 Jul 9(173). doi: 10.3791/62475.
The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.
如今，长非编码 RNA（lncRNA）在生理和病理生理学中的作用日益重要，因此通过鉴定其分子伴侣（DNA、蛋白质和/或 RNA）来描述其互作组至关重要。后者可以通过涉及蛋白质的网络与 lncRNA 相互作用，也可以参与直接的 RNA/RNA 相互作用。因此，我们开发了一种易于使用的 RNA 下拉程序，该程序允许使用补骨脂素鉴定与 lncRNA 发生直接 RNA/RNA 相互作用的 RNA，补骨脂素是一种仅交联 RNA/RNA 相互作用的分子。lncRNA 二级结构的生物信息学建模允许选择几个具有强亲和力的特异性反义 DNA 寡核苷酸探针，这些探针针对显示低内部碱基配对概率的区域。由于设计的特异性探针靶向 lncRNA 全长上的可及区域，因此可以在 lncRNA 的序列中描绘 RNA 相互作用区域。当与高通量 RNA 测序结合使用时，该方案可用于研究感兴趣的 lncRNA 的整个直接 RNA 互作组。
