Using CRISPR/Cas9 Forward Genetic Screens to Study Degradation Pathways at the Endoplasmic Reticulum

Intracellular protein quality control is essential to remove damaged or misfolded proteins and limit the cellular toxicity caused by protein aggregation, which is apparent in diseases such as Alzheimer's disease. Two main pathways are responsible for regulating protein turnover in cells, the ubiquitin proteasome system and autophagy. Transmembrane proteins, or those targeted to the secretory pathway are regulated by a specialised form of proteasomal degradation, endoplasmic reticulum associated degradation (ERAD). Whilst the ubiquitin enzymes involved in ERAD are well characterised, the quality control pathway for misfolded cytosolic proteins is less clear. Recently, using a mutagenesis forward genetic screen in near-haploid mammalian cells, the Nathan lab have shown that a model misfolded soluble protein, mCherry-CL1, is in fact degraded at the cytosolic face of the ER by two ER resident ubiquitin E3 ligases, MARCH6 and TRC8. Here, I have used genome-wide CRISPR/Cas9 forward genetic screens to search for new genes involved in protein quality control. Firstly, I developed a CRISPR/Cas9 forward genetic screen in HeLa cells expressing mCherry-CL1 to see if I could elucidate more components of this protein quality pathway. This identified UBE2J2 as the predominant E2 conjugating enzyme recruited by MARCH6 for degrading the CL1 degron. In addition, I have shown that UBE2J2 associates with MARCH6 and that this E2 enzyme is also required for the degradation of the endogenous MARCH6 substrate, squalene monooxygenase. Secondly, I have established a CRISPR/Cas9 forward genetic screen to explore the links between oxygen sensing and cholesterol synthesis to examine how hypoxia influences the ER quality control of HMG CoA reductase (HMGCR). In sterol deplete conditions, hypoxia promotes HMGCR degradation in a pathway which seems to be independent of the main regulators of the hypoxic response, the hypoxia inducible transcription factors (HIFs). In combination, I envisage that these genetic approaches will provide important insights into protein quality control and ERAD