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Analysis of the “moonlighting” proteins in the mTOR-signalling pathway

Periodic Reporting for period 1 - MoTOR (Analysis of the “moonlighting” proteins in the mTOR-signalling pathway)

Reporting period: 2016-02-01 to 2018-01-31

RNA is the language in which the genome talks. This smaller molecule carries pieces of messages to different effect places in the cell. The cell decorates the RNA molecules with numerous modifications and RNA binding proteins to regulate this information flow. The mTOR kinase is the control hub of metabolic regulation in the cell. The two complexes the mTOR kinase forms integrate the signal from the environment and inside the cell, make decisions, and communicate the decisions to other parts of the cell and thus influence a broad range of growth events. In general, the decisions that mTOR complexes take support cell growth and proliferation, and inhibit cell death. Impairment in these protein complexes is often observed in tumours, Alzheimer’s disease, and several metabolic disorders. Recent studies have shown that a series of mTOR related proteins were identified as non-canonical RNA binding proteins. This finding has shed light on the connection between the metabolic control hub mTOR kinase and the RNA molecules. What is striking though, unlike the classical RNA binding proteins, most of the proteins found here do not possess an RNA binding domain. Since the mechanism and biological function of this new interaction are unknown, these novel RNA binding proteins are named enigmRBPs (RNA binding proteins with enigmatic function).

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 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.
Work carried out in this project has allowed me to characterise the novel RNA binding protein Fpr1p. Although this project was carried out purely as a basic scientific discovery and driven by curiosity, the finding of it may result in a valuable product for the pharmaceutical industry at some stage in the future. During this project, I have identified a mutation that not only renders the protein inactive towards RNA but also changed the sensitivity of the strain to the immunosuppressant drug FK506. This finding together with the fact that Fpr1p interacts with RNA will provide insights to guide the discovery of new drugs.
The yeast system used to assess the RNA binding activity of Fpr1p
Point mutations introduced to the protein Fpr1p show different level of RNA binding activity