Postdoctoral Research Fellowship
Role of RNA and RNA-binding proteins in EGFR endocytosis and signaling
Background: Endocytosis is a critical regulator of EGFR signaling and downstream biological responses, including proliferation, migration, stem cell homeostasis, and differentiation. The EGFR can be internalized by different endocytic pathways that direct the receptor to different fates (e.g., recycling or degradation), influencing the strength, duration and spatiotemporal resolution of signaling (Sigismund et al., Dev Cell 2008; Sigismund et al., EMBO J 2013; Caldieri et al., Science 2017). Indeed, we found that, while clathrin-mediated endocytosis (CME) is mainly involved in EGFR recycling and sustaining signalling, EGFR internalization through a recently identified non-clathrin endocytosis (NCE) leads primarily to receptor degradation and signal extinction, representing a crucial safety mechanism to protect cells from overstimulation. A biochemical purification of NCE vesicles coupled to quantitative mass spectrometry and RNAi-based validation screening, led to the identification of a number of EGFR-NCE regulators, among which proteins belonging to endoplasmic reticulum (ER) and mitochondria. In agreement with this observation, we showed that internalization via NCE involves the formation of tripartite contact sites between the PM, the ER and the mitochondria. These contacts are established upon stimulation with high dose of EGF and their formation is mediated by the ER-resident protein Reticulon 3 (RTN3). Importantly, they are sites where EGF-dependent localized Ca2+ signalling occurs and propagates, possibly controlling cellular metabolic processes that extend beyond those associated with the canonical EGFR signalling pathway.
Among the regulatory proteins associated with NCE vesicles in our proteomics screening, there was a significant number of RNA-binding proteins with known roles in RNA metabolism and liquid-liquid phase separation of RNA-protein granules. In addition, preliminary data suggest that RNA itself could be associated with EGFR-NCE vesicles and functionally involved in the pathway. This discovery might open new perspectives into mechanism, regulation and function of EGFR-NCE, suggesting a possible cross talk between EGFR endocytosis and RNA scaffolding and/or metabolic functions.
Project aims: The successful candidate will be involved in:
- Identification of RNA molecules associated with EGFR-NCE (g., APEX-RNA coupled to RNAseq)
- Identification of RNA-binding proteins associated with EGFR-NCE (g., APEX-based proteomics)
- Localization and functional studies on selected RNA molecules and RNA-binding proteins (g., endocytosis, trafficking and signaling assays, EGF-dependent biological readouts, organoid morphogenetic and stem cell assays)
The research activity will be performed at the IEO in Milan, Italy, under the supervision of Prof. Sara Sigismund and Prof. Pier Paolo Di Fiore. The successful candidate will join a highly motivated research group and will have access to all the facilities, expertise, knowhow and tools available in the PIs’ groups and at the IEO.
Required skills and experience:
- PhD in Biology, Biotechnology or related fields;
- excellent English communication skills, both written and verbal.
Desirable skills and experience:
- experience with cell biology, molecular biology and biochemistry techniques;
- experience with RNA-based techniques;
- experience with cell and organoid cultures.
Salary will be commensurate to CV, previous experience and skills.
The work location is the IEO, Department of Experimental Oncology, Via Adamello 16, 20141 Milan.