title: RNA interference screens to uncover membrane protein biology.
abstract: In this review, we discuss the use of RNA interference screens to identify genes involved in the regulation and function of membrane proteins. Briefly, cells expressing the membrane protein of interest can be transduced with a pooled lentiviral short-hairpin RNA (shRNA) library containing tens of thousands of unique shRNAs. Transduced cells are then selected or fractionated based on specific critera, such as membrane protein expression or function. shRNAs from selected cell populations are then deconvoluted and quantified using microarray analyses or high-throughput sequencing technologies. This allows individual shRNAs to be scored and cutoffs can be made to generate a list of shRNA hits. Bioinformatic analyses of gene targets of shRNA hits can be used to identify pathways and processes associated with membrane protein biology. To illustrate this functional genomics approach, we discuss pooled lentiviral shRNA screens that were performed to identify genes that regulate the transcription and cell-surface expression of the cancer stem cell marker CD133. This approach can be adapted to study other membrane proteins, as well as specific aspects of membrane proteins, such as their function or downstream signaling effects.
authors.affiliation: Donnelly Centre and Banting and Best Department of Medical Research/Department of Molecular Genetics, University of Toronto, 830-160 College Street, Toronto, ON M5S 3E1, Canada. ab.mak@mail.utoronto.ca.
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Mak
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D034622:RNA Interference
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D014408:Biomarkers, Tumor; D006023:Glycoproteins; D008565:Membrane Proteins; C000606023:PROM1 protein, human; D010455:Peptides
keywords: AC133; CD133; RNA interference; high-throughput screening; membrane proteins; short hairpin RNA