title: Translational profiling through biotinylation of tagged ribosomes in zebrafish.
abstract: Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.
authors.affiliation: Department of Biochemistry & Biophysics, UCSF, San Francisco, CA 94158, USA michael.housley@mpi-bn.mpg.de didier.stainier@mpi-bn.mpg.de benoit.vanhollebeke@ulb.ac.be.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Housley
authors.affiliation: Department of Biochemistry & Biophysics, UCSF, San Francisco, CA 94158, USA.
authors.forename: Sven
authors.initials: S
authors.lastname: Reischauer
authors.affiliation: URBC-Narilis, University of Namur, Namur B-5000, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Dieu
authors.affiliation: URBC-Narilis, University of Namur, Namur B-5000, Belgium.
authors.forename: Martine
authors.initials: M
authors.lastname: Raes
authors.affiliation: Department of Biochemistry & Biophysics, UCSF, San Francisco, CA 94158, USA michael.housley@mpi-bn.mpg.de didier.stainier@mpi-bn.mpg.de benoit.vanhollebeke@ulb.ac.be.
authors.forename: Didier Y R
authors.initials: DY
authors.lastname: Stainier
authors.affiliation: Department of Biochemistry & Biophysics, UCSF, San Francisco, CA 94158, USA michael.housley@mpi-bn.mpg.de didier.stainier@mpi-bn.mpg.de benoit.vanhollebeke@ulb.ac.be.
authors.forename: Benoit
authors.initials: B
authors.lastname: Vanhollebeke
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019921:Biotinylation; D003066:Coenzyme A Ligases / Q000737:chemistry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000737:chemistry; D017403:In Situ Hybridization; D013058:Mass Spectrometry; D018482:Muscle, Skeletal / Q000473:pathology; D011132:Polyribosomes / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology*; D012269:Ribosomal Proteins / Q000502:physiology; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C000593368:Rpl10a protein, zebrafish; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D003066:Coenzyme A Ligases; C051127:biotin-CoA ligase
keywords: BirA; Gene expression profiling; Muscle development; TRAP