Periodic Reporting for period 1 - MoTOR (Analysis of the “moonlighting” proteins in the mTOR-signalling pathway)
Période du rapport: 2016-02-01 au 2018-01-31
My project “MoTOR” aims to answer the question, why the mTOR related proteins bind to RNA and what is the biological function of this interaction. Every novel interaction provides new drug target, the results of this work could provide insights to discover or design new drugs. The first objective was to validate the candidate RNA binding proteins. The second objective was to study the confirmed RNA binding proteins and their function. The third objective was using the Next Generation Sequencing method to identify the interacting RNA of these candidates.
The overall project has identified the ADP-ribosylation factor Arf1p and the FKBP12 homolog Fpr1p as bona fide RNA binding proteins. We further followed up Fpr1p and identified a point mutation in the protein that disrupts the interaction of Fpr1p to RNA. Interestingly, this mutation also displays various growth phenotype when the cell is treated with drugs. Using eCLIP, we could reveal that the species of RNA interacting Fpr1p is the transfer RNAs (tRNAs). Further functions of this protein will be studied in the future.
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.