Chakraborty Alina, Mukherjee Srimoyee, Saha Sucharita, De Soumasree, Sengupta Bandyopadhyay Sumita
Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India.
Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
Biochem J. 2017 Jul 3;474(14):2349-2363. doi: 10.1042/BCJ20170051.
Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human mRNA revealed several conserved sequences with conventional -acting elements. A 216 nucleotide containing proximal -element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein -factors interacting with mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent co-association of both these proteins with mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic mRNA and reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both mRNA and protein levels. Collectively, this report establishes the stabilization of mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal -element.
白血病抑制因子（LIF）是一种强大的多效细胞因子，参与多种生物学活动，因此需要对其表达进行精确的时空控制。本研究表明，在人组织细胞淋巴瘤细胞系U937中，LIF对佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）的反应增强，主要是通过其mRNA的稳定来实现的。对人LIF mRNA长3'非翻译区的功能表征揭示了几个具有传统顺式作用元件的保守序列。一个包含近端元件的216个核苷酸，有两个AUUUA五聚体和四个多聚rC序列，显示出显著的mRNA去稳定化潜力，在用PMA处理后，表现出稳定化活性。亲和层析，随后对PMA处理的U937提取物进行蛋白质免疫印迹和RNA共免疫沉淀，确定核仁素和PCBP1是与LIF mRNA相互作用的两种蛋白质因子，特别是与近端非传统富含AU的区域相互作用。PMA诱导核仁素和PCBP1的核质转运。在存在核糖核酸酶的情况下，共沉淀减少，证明了这两种蛋白质与LIF mRNA的RNA依赖性共结合。核仁素的异位过表达显示内源性LIF mRNA和报告基因均稳定，而核仁素和PCBP1的敲低显示LIF mRNA和蛋白质水平均显著降低。总体而言，本报告确定了PMA介导的LIF mRNA的稳定，这是由两种RNA结合蛋白核仁素和PCBP1与近端元件的相互作用介导的。
