Nucleolin is a second component of the CD154 mRNA stability complex that regulates mRNA turnover in activated T cells.
CD154 (CD40L) mRNA turnover is regulated in part at the posttranscriptional level by a protein complex (termed Complex I) that binds to a highly CU-rich region of the 3'UTR. Polypyrimidine tract-binding protein (PTB) has previously been identified as a major RNA-binding protein in Complex I. Nondenaturing gel filtration of total extract from Jurkat T cells demonstrated that the CD154 mRNA-binding activity migrates as a approximately 200-kDa complex, indicating the presence of multiple complex-associated proteins. We have currently undertaken a biochemical approach to further characterize Complex I and observed that it segregates over DEAE-Sepharose into two subcomplexes (termed I-L and I-U). Furthermore, nucleolin was identified as a component of both subcomplexes and was shown that it is the major RNA-binding protein in I-U. To directly demonstrate the biological significance of Complex I binding to the CD154 transcript, cytoplasm from human Jurkat cells was fractionated over a sucrose gradient and the different cellular fractions subjected to immunoprecipitation with anti-PTB and anti-nucleolin Abs. RT-PCR of the immunoprecipitated products using CD154-specific primers clearly demonstrated that nucleolin and PTB are associated with CD154 mRNA in both the ribonucleoprotein and polysome fractions. These data strongly support a model whereby nucleolin and PTB are integral to the stability of CD154 mRNA and are components of the CD154 ribonucleoprotein particle associated with actively translating ribosomes.
Singh
Karnail
K
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA.
Laughlin
Jennifer
J
Kosinski
Penelope A
PA
Covey
Lori R
LR
D023201:CD40 Ligand / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D046911:Macromolecular Substances; D010750:Phosphoproteins / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D000097796:Nucleolin
D023201:CD40 Ligand; D046911:Macromolecular Substances; D010750:Phosphoproteins; D038941:Polypyrimidine Tract-Binding Protein; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000097796:Nucleolin