Microbial iCLIP2: Enhanced mapping of RNA-protein interaction by promoting protein and RNA stability

From synthesis to decay, the entire lifecycle of RNAs is precisely regulated by RNA-binding proteins (RBPs). Understanding the RBP-RNA interaction network, specifically their binding position on the transcriptome, is crucial for uncovering the regulatory potential of these proteins. CLIP-based methods, particularly the iCLIP method, represent a state-of-the-art technique for identifying RBP binding sites at single-nucleotide resolution. However, these methods require adaption for microbiological applications. Here, we have optimized the recent iCLIP2 method using the RBP Rrm4 for use in the fungal model system Ustilago maydis and successfully demonstrated its efficacy in conditions with high RNAse and protease activity. Overall design: Optimizing iCLIP2 for detecting protein-RNA interactions in microbial conditions using the fungal model system Ustilago maydisRNA结合蛋白（RNA-binding proteins, RBPs）精准调控RNA从合成到降解的完整生命周期。解析RBP-RNA互作网络，尤其是其在转录组上的结合位点，对于揭示这类蛋白质的调控潜能至关重要。基于CLIP的实验方法，尤其是iCLIP技术，是当前在单核苷酸分辨率下鉴定RBP结合位点的顶尖技术。然而，这类方法尚需适配微生物学研究场景。本研究以真菌模式系统玉米黑粉菌（Ustilago maydis）中的RNA结合蛋白Rrm4为对象，对最新的iCLIP2方法进行了优化，并在高核糖核酸酶（RNAse）与蛋白酶活性的实验条件下成功验证了该方法的有效性。实验设计概述：以玉米黑粉菌（Ustilago maydis）为真菌模式系统，优化iCLIP2技术以检测微生物环境中的蛋白质-RNA互作。