title: Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).
abstract: RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA.
authors.affiliation: Department of Biological Sciences, University of Texas at Dallas, Richardson, TX 75080, United States.
authors.forename: Tzu-Fang
authors.initials: TF
authors.lastname: Lou
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, United States.
authors.forename: Chase A
authors.initials: CA
authors.lastname: Weidmann
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, United States.
authors.forename: Jordan
authors.initials: J
authors.lastname: Killingsworth
authors.affiliation: Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, United States.
authors.forename: Traci M
authors.initials: TM
authors.lastname: Tanaka Hall
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, United States; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States.
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Goldstrohm
authors.affiliation: Department of Biological Sciences, University of Texas at Dallas, Richardson, TX 75080, United States. Electronic address: zachary.campbell@utdallas.edu.
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Campbell
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D052156:SELEX Aptamer Technique; D017423:Sequence Analysis, RNA / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D029721:Drosophila Proteins; D016601:RNA-Binding Proteins; D014764:Viral Proteins; C074333:pum protein, Drosophila; C070574:nos protein, Drosophila; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Combinatorial control; NOS; Pumilio; RNA; SELEX; Sequencing