TitleModulation of CD40 ligand (CD40L) expression by polypyrimidine tract-binding protein
NameMatus Nicodemos, Rodrigo (author), Covey, Lori R (chair), Kiledjian, Mergerditch (internal member), Xie, Ping (internal member), Rutgers University, Graduate School - New Brunswick,
SubjectMicrobiology and Molecular Genetics,
DescriptionCD40 ligand (CD40L or CD154) is a protein expressed on activated CD4+ T cells, which is crucial for antibody-dependent and cell-mediated immunity. The expression of CD40L is tightly regulated at multiple levels throughout a time course of T cell activation. At the post-transcriptional level the CD40L message is rapidly degraded at early time points of activation followed by a significant increase in message stability at later times of activation (24-48 hr). Previous work from our lab revealed that a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3'UTR at later times of T cell activation. To understand the direct relationship between PTB and CD40L mRNA stability and subsequently CD40L expression, we used viral RNA interference against PTB and scrambled shRNA (Control) sequence in model CD40L mRNA stability T cell line, Jurkat/D1.1. Downregulation of PTB resulted in dramatic decrease in half-life of the CD40L mRNA. The downregulation of PTB did not significantly change the percentage of CD40L+ cells, but caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Cellular fractionation of CD40L mRNA from shCTRL- and shPTB-infected cells revealed a novel role for nuclear PTB in retaining the CD40L mRNA in the nucleus. In addition, cytoplasmic PTB is important for optimal association of CD40L message with translating polysomes. Analysis of PTB cellular distribution during a time course of CD4+ T cell activation revealed cytoplasmic and nuclear localization in all resting and activated cells. However, there was an increase in cytoplasmic PTB expression at late times of activation. Binding studies revealed that CD40L mRNA is bound by nuclear PTB at all times of activation indicating that the requirements for binding of CD40L message by nuclear versus cytoplasmic PTB is highly distinct. Finally, the binding of CD40L message corresponded to a post-translational modification of cytoplasmic PTB that appears to correlate with a change in phosphorylation status of PTB. Confocal microscopy analysis of CD4+ T cells with activation-induced CD40L mRNA stability revealed co-localization of PTB and CD40L mRNA at distinct foci, suggesting a role of PTB in the localization of the stable CD40L message during T cell activation.
NoteIncludes bibliographical references
Noteby Rodrigo A. Matus Nicodemos
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.