Discovery of a novel repeated RNA-binding motif conserved in diverse uncharacterized proteins of photosynthetic organisms

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The plant-specific factor HCF145 was shown to specifically protect the plastid psaA-psaB-rps14 mRNA against 5 - 3 exonucleolytic attack via specific binding of the newly defined transcript-binding motif repeat (TMR) domains to its 5 UTR, 52 nucleotides downstream of the transcription start site. TMR motifs are conserved in quite diverse, so far uncharacterized proteins in Physcomitrella patens, red and green algae as well as in the cyanobacterium Microcoleus sp. PCC 7113. Representative TMR proteins in the four lineages could be obtained in high purity and solubility and bind heterologous ssRNA with high affinity and specificity, indicating that TMR motifs extend the repertoire of RNA-binding domain families that emerged in photosynthetic organisms. Remarkably, besides PPR, OPR and PUF proteins, TMR proteins represent a novel and quite interesting class of RNA-binding proteins with multiple RNA recognition motifs, also proposed to form a regular superhelical structure. Presumably, TMR motifs also provide a combinatorial amino acid code for RNA recognition. This project is a pioneer work on the RNA-binding function of representative members of the previous unrecognized TMR family in photosynthetic organisms. We will first focus on the function and the precise RNA targets of TMR proteins in vivo by using tandem affinity purification-tagging strategies in transgenic organisms and co-immunoprecipitation in combination with RNA-seq (RIP-seq). Data will be complememted by mutant analysis as well as in vitro RNA-binding and structural studies. In parallel, immunoprecipitates will be analysed by mass spectrometry to gain first informations about TMR interaction partners in the respective organisms.