genome-wide-rnai-screening-to-identify-host-factors-that-modulate

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Children's Hospital of Eastern Ontario (CHEO) Research Institute

PINK1/PARK6 and Parkin/PARK2 are amongst the most commonly mutated genes associated with recessive forms of familial Parkinson's disease. Recent evidence indicates that the proteins they encode, PINK1 and Parkin, function in the same pathway to mediate the selective autophagic clearance of dysfunctional mitochondria. Upon mitochondrial damage, PINK1 is stabilized on the outer mitochondrial membrane where it phosphorylates ubiquitin, generating a signal for the recruitment and activation of Parkin. However, key mechanistic questions still exist regarding Parkin recruitment, including whether or not other factors are required for the PINK1 and Parkin pathway. We describe a method below using high-throughput RNA interference technology to interrogate the genome for novel components of the PINK1 and Parkin pathway.

High-content functional genomic screening to identify novel regulators of the PINK1-Parkin pathway.

Programmed cell death 4 (PDCD4) is a tumour suppressor implicated in cancer development and progression and was recently identified as a repressor of cap-independent translation of specific genes involved in the regulation of apoptosis. We show that the RNA-binding protein HuR binds to the PDCD4 3'UTR to protect it from miR-21-induced silencing. However, following H2O2 treatment, PDCD4 mRNA is degraded via miR-21 binding. Importantly, we identify HuR as a novel substrate of the ERK8 kinase pathway in response to H2O2 treatment. We show that phosphorylation of HuR by ERK8 prevents it from binding to PDCD4 mRNA and allows miR-21-mediated degradation of PDCD4.

ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism.

SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known.

Article	Year
High-content functional genomic screening to identify novel regulators of the PINK1-Parkin pathway. Methods in enzymology\| PubMed ID: 25416349	2014
ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism. Oncotarget\| PubMed ID: 26595526	2016
Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts. Orphanet journal of rare diseases\| PubMed ID: 27317422	2016

Stephen D. Baird

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