Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
Nat Commun. 2022 May 17;13(1):2719. doi: 10.1038/s41467-022-30284-w.
Photo-induced cross-linking is a mainstay technique to characterize RNA-protein interactions. However, UV-induced cross-linking between RNA and proteins at "zero-distance" is poorly understood. Here, we investigate cross-linking of the RBFOX alternative splicing factor with its hepta-ribonucleotide binding element as a model system. We examine the influence of nucleobase, nucleotide position and amino acid composition using CLIR-MS technology (crosslinking-of-isotope-labelled-RNA-and-tandem-mass-spectrometry), that locates cross-links on RNA and protein with site-specific resolution. Surprisingly, cross-linking occurs only at nucleotides that are π-stacked to phenylalanines. Notably, this π-stacking interaction is also necessary for the amino-acids flanking phenylalanines to partake in UV-cross-linking. We confirmed these observations in several published datasets where cross-linking sites could be mapped to a high resolution structure. We hypothesize that π-stacking to aromatic amino acids activates cross-linking in RNA-protein complexes, whereafter nucleotide and peptide radicals recombine. These findings will facilitate interpretation of cross-linking data from structural studies and from genome-wide datasets generated using CLIP (cross-linking-and-immunoprecipitation) methods.
光诱导交联是一种用于表征 RNA-蛋白质相互作用的主要技术。然而，RNA 和蛋白质之间在“零距离”下的 UV 诱导交联还不太清楚。在这里，我们以 RBFOX 可变剪接因子与其七核苷酸结合元件的交联为例进行研究。我们使用 CLIR-MS 技术（同位素标记 RNA 的交联和串联质谱）来研究核碱基、核苷酸位置和氨基酸组成的影响，该技术具有在 RNA 和蛋白质上进行位点特异性解析的能力。令人惊讶的是，交联仅发生在与苯丙氨酸π堆积的核苷酸上。值得注意的是，这种π堆积相互作用对于侧翼苯丙氨酸的氨基酸参与 UV 交联也是必需的。我们在几个已发表的数据集的研究中证实了这些观察结果，其中可以将交联位点映射到高分辨率结构上。我们假设与芳香族氨基酸的π堆积会激活 RNA-蛋白质复合物中的交联，随后核苷酸和肽自由基会重新组合。这些发现将有助于解释结构研究和使用 CLIP（交联和免疫沉淀）方法生成的全基因组数据集的交联数据。
