The first project objective was to validate the RNA binding proteins identified by previous studies. This objective was accomplished by combing UV-crosslinking, immunoprecipitation, and radioactive labelling. Briefly, the RNA molecules that interact with the protein of interest are immobilised to the protein by UV-crosslinking. Then the resulted RNA-protein complex was enriched using an antibody against the protein of interest. To visualise, RNA was radioactively labelled by polynucleotide kinase, and the RNA-protein complex separated by an SDS-PAGE. This procedure proved to be quite challenging due to the nature of the proteins we are interested in. Unlike the classical RNA binding proteins, the enigmRBPs bind to RNA weakly. Among all the candidates tested, we could confirm the ADP-ribosylation factor Arf1p and the FK506 binding protein Fpr1p as positive candidates. We decided to focus the work on Fpr1p after that given the intriguing biological function of Fpr1p. Fpr1p and its human homolog FKBP12 is known to be involved in autoimmune disorders, mTOR inhibition, and intracellular calcium regulation. In the following experiments, We could further identify the peptide that interacts with RNA. By introducing a single amino acid point mutation in this peptide, Fpr1p loses its ability to interact with RNA.
The second objective was to analyse the function of the RNA interaction. We have characterised the growth phenotype, mTORC1 activity, and response to drug treatment. We could observe that the RNA binding deficient mutant FPR1F94V compared to the wild-type strain showed a slow growth phenotype, similar to the FPR1 knockout strain. Since Fpr1p can bind to rapamycin and relocates to and inhibits the mTORC1 complex, it is interesting to test if Fpr1F94V mutant also does so. The mTORC1 activity was read out by probing the phosphorylation state of its substrates. As expected, Fpr1F94V behaved like its wild-type counterpart and inhibits mTORC1 under rapamycin treatment. This result indicates that the RNA binding mutants have similar affinity to rapamycin in vivo. Fpr1p also binds to other drugs, and the most interesting one is FK506. Binding of FK506 to the human homolog of Fpr1p leads to the interference of T-cell activation, so that inhibit autoimmune disorder. As expected, FK506 treatment did not affect the growth of the knockout strain and inhibits the growth of FPR1 wild-type. However, to our surprise, the RNA binding mutants Fpr1F94V also did not respond to FK506 treatment.
The third objective of the action was to identify the RNAs that are bound by Fpr1p. We have accomplished this by applying a modified version of the eCLIP method. eCLIP has given us a comprehensive view of the RNAs interacting Fpr1p and facilitated the characterisation of Fpr1p as an enigmRBP. Interestingly, the primary targets of Fpr1p are the transfer RNAs. This finding has added new aspects of our understanding about the FK506 binding protein Fpr1p.
