Matus-Nicodemos Rodrigo, Vavassori Stefano, Castro-Faix Moraima, Valentin-Acevedo Anibal, Singh Karnail, Marcelli Valentina, Covey Lori R
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA.
J Immunol. 2011 Feb 15;186(4):2164-71. doi: 10.4049/jimmunol.1003236. Epub 2011 Jan 17.
CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3' untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.
CD40L（CD154）在转录后水平受激活诱导过程调控，该过程导致T细胞激活较长时间后转录本高度稳定。转录本稳定性由含聚嘧啶序列结合蛋白（PTB）的复合物（复合物I和II）介导，这些复合物与3'非翻译区内三个相邻的富含CU的序列结合。为了评估PTB在CD40L mRNA表达和分布中的作用，在原代T细胞和一个重现受调控的CD40L mRNA衰变稳定期的T细胞系中，使用短发夹RNA靶向PTB。PTB敲低导致mRNA稳定性显著降低，进而导致CD40L表面表达降低。PTB对于CD40L mRNA在细胞核与细胞质之间以及细胞质中胞质溶胶与翻译多核糖体之间的适当分布也至关重要。仅在细胞质而非细胞核的PTB中观察到激活诱导的PTB特异性核糖核蛋白复合物的形成，这表明由细胞定位定义的蛋白质存在功能差异。最后，我们观察到细胞质和细胞核PTB异构体彼此之间存在差异修饰，并且细胞质PTB的变化与激活诱导的磷酸化一致。这项工作共同表明，差异修饰的PTB通过以下方式在多个步骤调节CD40L表达：1）将CD40L mRNA保留在细胞核中；2）在激活后期直接调节mRNA稳定性；3）形成优先与翻译核糖体结合的核糖核复合物，从而导致CD40L蛋白水平升高。
