title: Synthetic antibodies as tools to probe RNA-binding protein function.
abstract: RNA-binding proteins (RBPs) have essential roles in post-transcriptional regulation of gene expression. They bind sequence elements in specific mRNAs and control their splicing, transport, localization, translation, and stability. A complete understanding of RBP function requires identification of the target RNAs that an RBP regulates, the mechanisms by which the RBP regulates these targets, and the biological consequences for the cell in which these transactions occur. Antibodies are key tools in such studies: first, mRNA targets of RBPs can be identified by co-immunoprecipitation of RBPs with their associated RNAs followed by microarray analysis or sequencing; second, partner proteins can be identified by immunoprecipitation of the RBP followed by mass spectrometry; third, the mechanisms and functions of RBPs can be inferred from loss-of-function studies employing antibodies that block RBP-RNA interactions. One potentially powerful approach to making antibodies for such studies is the generation of synthetic antibodies using phage display, which involves in vitro selection using a human-designed antibody library to generate antibodies that recognize a target protein. Using two well-characterized Drosophila RNA-binding proteins, Staufen and Smaug, for proof-of-principle, we demonstrate that synthetic antibodies can be generated and used either to perform RNA-coimmunoprecipitations (RIPs) to identify RBP-bound mRNAs, or to block RBP-RNA interactions. Given that synthetic antibody selection protocols are amenable to high-throughput antibody production, these results demonstrate that synthetic antibodies can be powerful tools for genome-wide studies of RBP function.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: John D
authors.initials: JD
authors.lastname: Laver
authors.forename: Kristin
authors.initials: K
authors.lastname: Ancevicius
authors.forename: Pietro
authors.initials: P
authors.lastname: Sollazzo
authors.forename: J Timothy
authors.initials: JT
authors.lastname: Westwood
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Howard D
authors.initials: HD
authors.lastname: Lipshitz
authors.forename: Craig A
authors.initials: CA
authors.lastname: Smibert
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D000941:Antigens / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D000906:Antibodies; D000941:Antigens; D029721:Drosophila Proteins; D015335:Molecular Probes; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C402587:smg protein, Drosophila; C069927:stau protein, Drosophila