Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain.
Centre de Recerca en Agrigenòmica, Consortium CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, 08193 Barcelona, Spain.
Methods. 2020 Jun 1;178:19-32. doi: 10.1016/j.ymeth.2019.09.002. Epub 2019 Sep 4.
Understanding which proteins and RNAs directly interact is crucial for revealing cellular mechanisms of gene regulation. Efficient methods allowing to detect RNA-protein interactions and dissect the underlying molecular origin for RNA-binding protein (RBP) specificity are in high demand. The recently developed recombination-Y3H screening (rec-Y3H) enabled many-by-many detection of interactions between pools of proteins and RNA fragments for the first time. Here, we test different conditions for protein-RNA interaction selection during rec-Y3H screening and provide information on the screen performance in several selection media. We further show that rec-Y3H can detect the nucleotide and amino acid sequence determinants of protein-RNA interactions by mutating residues of interacting proteins and RNAs simultaneously. We envision that systematic RNA-protein interface mutation screening will be useful to understand the molecular origin of RBP selectivity and to engineer RBPs with targeted specificities in the future.
了解哪些蛋白质和 RNA 直接相互作用对于揭示基因调控的细胞机制至关重要。高效的方法可以检测 RNA-蛋白质相互作用，并剖析 RNA 结合蛋白 (RBP) 特异性的潜在分子起源，因此这些方法的需求很高。最近开发的重组 Y3H 筛选 (rec-Y3H) 首次允许在蛋白质和 RNA 片段库之间进行多次相互作用检测。在这里，我们在 rec-Y3H 筛选过程中测试了不同的蛋白质-RNA 相互作用选择条件，并提供了有关几种选择培养基中筛选性能的信息。我们进一步表明，rec-Y3H 可以通过同时突变相互作用的蛋白质和 RNA 的残基来检测蛋白质-RNA 相互作用的核苷酸和氨基酸序列决定因素。我们设想，系统的 RNA-蛋白质界面突变筛选将有助于理解 RBP 选择性的分子起源，并在未来设计具有靶向特异性的 RBP。
