title: Isolation and analyses of axonal ribonucleoprotein complexes.
abstract: Cytoskeleton-dependent RNA transport and local translation in axons are gaining increased attention as key processes in the maintenance and functioning of neurons. Specific axonal transcripts have been found to play roles in many aspects of axonal physiology including axon guidance, axon survival, axon to soma communication, injury response and regeneration. This axonal transcriptome requires long-range transport that is achieved by motor proteins carrying transcripts as messenger ribonucleoprotein (mRNP) complexes along microtubules. Other than transport, the mRNP complex plays a major role in the generation, maintenance, and regulation of the axonal transcriptome. Identification of axonal RNA-binding proteins (RBPs) and analyses of the dynamics of their mRNPs are of high interest to the field. Here, we describe methods for the study of interactions between RNA and proteins in axons. First, we describe a protocol for identifying binding proteins for an RNA of interest by using RNA affinity chromatography. Subsequently, we discuss immunoprecipitation (IP) methods allowing the dissection of protein-RNA and protein-protein interactions in mRNPs under various physiological conditions.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Ella
authors.initials: E
authors.lastname: Doron-Mandel
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Alber
authors.affiliation: Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Oses
authors.affiliation: Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA.
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.affiliation: Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA.
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Mike
authors.initials: M
authors.lastname: Fainzilber
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia, SC, USA.
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Twiss
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia, SC, USA.
authors.forename: Seung Joon
authors.initials: SJ
authors.lastname: Lee
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D034443:RNA Transport / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification*; D012584:Sciatic Nerve / Q000166:cytology*; D059467:Transcriptome / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein
keywords: Axon transport; Proteomics; RNA-binding protein; RNP; Sciatic nerve