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 maydis