Target mRNAs and binding sites of the key RNA-binding protein Rrm4 during microtubule-dependent mRNA transport in the plant pathogen Ustilago maydis

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Microtubule-dependent mRNA transport is important to determine the correct spatio-temporal protein expression. Crucial players in this process are RNA-binding proteins (RBPs) that recognize RNA zipcodes in target mRNAs. In order to understand the underlying biological mechanisms a transcriptome-wide view is of great value. In the phytopathogenic fungus Ustilago maydis, microtubule-dependent mRNA transport is essential during efficient growth of infectious filaments. Recent studies uncovered the key RNA-binding protein of this transport process. Rrm4 belongs to the group of ELAV-type RBPs containing three RNA recognition motives (RRM1-3). Rrm4 function involves recognition of cytotopically related target mRNAs. The underlying long distance transport of mRNAs is coupled to endosomal shuttling mediated by the minus-end and plus-end directed motors split Dyn1/2 and Kin3, respectively. Here, we plan to use new iCLIP method (individual nucleotide cross-linking and immune precipitation) for the transcriptome-wide identification of target mRNAs of Rrm4 as well as its binding sites at single nucleotide resolution. Combining data from different mutants and related homologous proteins will allow pinpointing the consensus binding sites for tandem RRM1/2 and RRM3 domains, which show functional differences. In a complementary approach, we will establish a universal research strategy that addresses the functions of novel proteins whose spatio-termporal expression is regulated by microtubule-dependent mRNA transport. To this end we will analyse respective deletion mutants as well as the influence of target mRNA transport on subcellular localization of encoded proteins. Next, we will use genetic and protein interaction approaches to gain more specific information on the function of these novel proteins. The described approaches will considerably improve our understanding of cell polarity, membrane trafficking, secretion, infection and biotechnology.