Shanghai Institute of Thoracic Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
College of Health Science and Technology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Methods Mol Biol. 2023;2712:29-43. doi: 10.1007/978-1-0716-3433-2_4.
Ferroptosis is a regulatory cell death process that is accompanied by large amounts of iron ion accumulation and lipid peroxidation. Photoactivated ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) is a method used to identify the binding sites of RNA-binding proteins (RBPs) on target RNAs with high resolution at the nucleotide level. By inserting photosensitive ribonucleoside analogs into new RNA transcripts of living cells, characteristic mutations can be generated during reverse transcription and be used to accurately locate the crosslinking position of RNAs and RBPs. The use of PAR-CLIP to detect interactions and determine precise crosslinking sites between RNAs and RBPs, or to search for RNAs upstream or downstream of ferroptosis pathways genes through known proteins, can help to clarify and verify the occurrence and regulation mechanisms of the various signaling pathways of ferroptosis. Furthermore, it may reveal new targets for ferroptosis detection and improve the treatment efficiency of ferroptosis-related diseases such as cancer and neurodegenerative diseases. Here, we introduce a specific PAR-CLIP protocol for monitoring the ferroptosis process.
铁死亡是一种伴随着大量铁离子积累和脂质过氧化的调节性细胞死亡过程。光激活核苷酸增强交联和免疫沉淀（PAR-CLIP）是一种用于在核苷酸水平上以高分辨率鉴定 RNA 结合蛋白（RBP）在靶 RNA 上结合位点的方法。通过将光敏核苷酸类似物插入活细胞中新的 RNA 转录本中，在逆转录过程中可以产生特征性突变，并用于准确定位 RNA 和 RBP 的交联位置。使用 PAR-CLIP 来检测 RNA 和 RBP 之间的相互作用，并确定其精确交联位点，或者通过已知蛋白在铁死亡途径基因的上下游搜索 RNA，有助于阐明和验证铁死亡各种信号通路的发生和调控机制。此外，它可能揭示铁死亡检测的新靶点，并提高癌症和神经退行性疾病等与铁死亡相关疾病的治疗效率。在这里，我们介绍了一种用于监测铁死亡过程的特定 PAR-CLIP 方案。
