title: Intracellular localization and interaction of mRNA binding proteins as detected by FRET.
abstract: BACKGROUND
  A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.
  
  
  RESULTS
  All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1.
  
  
  CONCLUSIONS
  Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.
authors.affiliation: Department of Medicine/Cardiology and Pharmacology, University of Colorado School of Medicine, 12700 East 19th Avenue, Aurora, CO 80045, USA. david.port@ucdenver.edu
authors.forename: Pamela S
authors.initials: PS
authors.lastname: David Gerecht
authors.forename: Molly A
authors.initials: MA
authors.lastname: Taylor
authors.forename: J David
authors.initials: JD
authors.lastname: Port
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization; D021381:Protein Transport / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D046912:Multiprotein Complexes; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins