title: Functional characterization of the HuR:CD83 mRNA interaction.
abstract: Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1-3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein:RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway.
authors.affiliation: Department of Cell Biology and Virology, Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Pieper
authors.forename: Susann
authors.initials: S
authors.lastname: Schirmer
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Prechtel
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Kehlenbach
authors.forename: Joachim
authors.initials: J
authors.lastname: Hauber
authors.forename: Jan
authors.initials: J
authors.lastname: Chemnitz
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers; D051959:ELAV Proteins / Q000235:genetics*; D006801:Humans; D008213:Lymphocyte Activation; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D017931:DNA Primers; D051959:ELAV Proteins; D012333:RNA, Messenger; D051997:ADP-ribosyl Cyclase 1