A complex containing polypyrimidine tract-binding protein is involved in regulating the stability of CD40 ligand (CD154) mRNA.
CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of "regulated instability" of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3' untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Delta1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Delta1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.
Kosinski
Penelope A
PA
Department of Cell Biology and Neuroscience, Rutgers, State University of New Jersey, Piscataway 08854, USA.
Laughlin
Jennifer
J
Singh
Karnail
K
Covey
Lori R
LR
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023201:CD40 Ligand / Q000235:genetics* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D038941:Polypyrimidine Tract-Binding Protein / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology; D017384:Sequence Deletion
D020413:3' Untranslated Regions; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; C450006:PTBP1 protein, human; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein; D023201:CD40 Ligand; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases