Microbial iCLIP2: enhanced mapping of RNA–protein interaction by promoting protein and RNA stability

The entire RNA life cycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV cross-linking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP-binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus <i>Ustilago maydis</i> as an example. Key challenges, such as inherently high RNase and protease activity in fungi, were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of cross-link events and improved the identification of Rrm4-binding sites. Thereby, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complexes I, III, and V-revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study using <i>U. maydis</i> as an example might serve as a blueprint for optimizing iCLIP2 procedures in other microorganisms with high RNase/protease conditions.