Die Identifikation von Ziel-Transkripten des RNA bindenden Proteins Rrm4 aus Ustilago maydis

Establishing an axis of polarity is a prerequisite for filamentous growth in fungi. Formation and maintenance of the underlying molecular gradients is often supported by active subcellular transport along the microtubule cytoskeleton conveying cargo such as proteins, vesicles or mRNA. In Ustilago maydis, the putative RNA-binding protein Rrm4 forms particles that move bidirectionally along the microtubule cytoskeleton within the fungal filament. The transport process itself as well as the RNA recognition motives (RRMs) within Rrm4 are important for accurate establishment of the axis of polarity and pathogenicity of the fungus. The aim of this work was to identify transcripts that are bound by Rrm4 within filaments. Applying CLIP (ultraviolet crosslinking and immunoprecipitation) it could be shown that Rrm4 interacted with RNA in vivo. Binding was mediated by the RRM domains of the protein. After purification, 55 transcripts could be identified that were specifically bound by Rrm4. A bioinformatic analysis revealed a CA-rich motif as potential sequence-specific binding site. Interestingly, part of the target transcripts encoded for proteins whose homologues are involved in polar growth in other organisms. To study the subcellular localisation of target transcripts, FISH (fluorescence in situ hybridisation) was established for U. maydis filaments. Quantification of the signal distribution was carried out using the therefore implemented program PIA (peak-identifying algorithm). For the mRNA of septin Cdc3, it could be demonstrated that it accumulated in particles and that formation of these particles depended on Rrm4. Rrm4-mediated distribution of these particles might be important to supply regions within the filament that are farther away from the nucleus. Accordingly, loss of mRNA particles in rrm4 deletion strains correlated with a strongly reduced accumulation of the Cdc3 protein within the hyphal tip. Taken together, these results indicate that Rrm4-mediated transport of the cdc3 mRNA might be critical for correct distribution of the encoded protein within the hyphae. By localising this and possible additional polarity factors, Rrm4-mediated posttranscriptional regulation could exert a regulatory function on polar growth.