Abstract
RNA-binding proteins (RBPs) utilize multiple RNA-binding domains (RBDs) to engage with extensive messenger RNA (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 the third RRM domain, however, they are not critical for function. Conversely, functionally essential binding sites are recognized by a more complex RBD interplay, strongly depending on RRM1 and RRM2. 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.