The entire RNA lifecycle, 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 crosslinking 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 Ustilago maydis 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 crosslink events and improved the identification of Rrm4 binding sites. Thus, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complex I, III and V - revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study serves as a paradigm for optimizing iCLIP2 procedures in challenging organisms or tissues under high RNase/ protease conditions.

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