David Månsson:
RNAi screening for characterisation of ER-associated degradation pathways in mammalian cells

Date: 15-09-2013    Supervisor: Lars Ellgaard

It is estimated that one third of all synthesized proteins in mammalian cells traverse the secretory pathway. Folding of proteins in the ER on their way to secretion is highly regulated. Proteins that are unable to achieve their native conformation are degraded by the ubiquitin-proteasome system in a process termed ER-associated degradation (ERAD). This mechanism proceeds through four steps involving recognition, dislocation, ubiquitination and proteasomal degradation. This report describes a high-throughput screening method for identification of hitherto unknown pathways for degradation. We present fluorescence-based RNAi screens in mammalian cells on TCR-α-GFP and HANSκLC, for identification of ERAD pathways. By validating the obtained screening hits we concluded that UBE2J2 is involved in TCR-α-GFP degradation, possibly by ubiquitination of C-terminal serine residues in TCR-α-GFP. Additionally, we also observed a stabilisation of HA-NSκLC after combined downregulation of UBEJ1 and UBE2E2.