Functional analysis of a tripartite stability element within the CD40 ligand 3' untranslated region.
We previously identified a cis-acting element within the 3' untranslated region of CD40 ligand messenger RNA (mRNA) that is composed of three complex binding sites and acts to increase mRNA stability in both in vitro and in vivo systems. We now demonstrate the functional consequences of the three binding sites with respect to increasing both luciferase activity and mRNA stability in a heterologous transcript expressed in a T-cell line. The internal region B was shown to be a bona fide stability element because its presence increased luciferase activity fourfold over the unmodified transcript and its removal from the XbaI-HaeIII region resulted in rapid degradation of the transcript. Region A contained both a binding site for a polypyrimidine-tract-binding protein (PTB)-mediated complex (Complex I) and an upstream, adjacent sequence that was a negative regulator of mRNA stability. Region C bound Complex II, which contained both PTB and heterogeneous nuclear ribonucleoproteinL (hnRNPL), and was less effective as a stability element on its own compared to region B. Our findings demonstrate differential levels of activity for the three binding sites relative to the turnover of CD40 ligand mRNA, suggesting that the lack of binding of Complex I/II during the early stages of T-cell activation contributes to the rapid degradation of the CD40 ligand mRNA transcript.
Laughlin
Jennifer
J
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA.
Oghlidos
Sanaz
S
Porter
Joseph F
JF
Matus-Nicodemos
Rodrigo
R
Sinquett
Frank L
FL
Marcelli
Valentina
V
Covey
Lori R
LR
D020413:3' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023201:CD40 Ligand / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein; D023201:CD40 Ligand; D049410:Luciferases, Renilla