Stoffel, N. K. | Search bioRxiv

bioRxiv2025

Dissecting the RNA binding capacity of the multi-RRM protein Rrm4 essential for endosomal mRNA transport

RNA-binding proteins (RBPs) utilize multiple RNA-binding domains (RBDs) to engage with extensive mRNA networks. Understanding the intricate interplay of modular RBDs is essential for uncovering RBP function. Yet, how individual RBDs shape transcriptome-wide interactions remains poorly understood. Here, we dissect the roles of the three RNA recognition motifs (RRMs) in the endosomal mRNA transporter Rrm4 during polar growth of Ustilago maydis. Using a comparative mutant-based iCLIP2 approach, we disclose an extensive inventory of RRM-specific binding sites. Most binding sites are prominently governed by RRM3, however, they are not critical for function. Conversely, functionally essential binding sites are recognized by a more complex RBD interplay, involving RRM1 and/or RRM2 with partial support from RRM3. By integrating transcriptome-wide RNA binding data with transcriptomics, we pinpoint their function as regulatory RNA elements affecting mRNA abundance, linking endosomal transport to stability. The modular RNA binding of Rrm4 defines distinct RNA regulons controlling mitochondrial activity, polarity factors, and cell wall remodeling - processes critical for polar growth. These findings disclose the intricate binding modes of an RBP in vivo, emphasizing how multiple RBDs differentiate functional binding sites from accessory ones to determine mRNA fate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/636894v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1af77b6org.highwire.dtl.DTLVardef@dc7178org.highwire.dtl.DTLVardef@9d7ebforg.highwire.dtl.DTLVardef@1ec5a05_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

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

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.

molecular biology↗