Lewis Stephen M, Veyrier Anne, Hosszu Ungureanu Nicoleta, Bonnal Sophie, Vagner Stéphan, Holcik Martin
Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, K1H 8L1, Canada.
Mol Biol Cell. 2007 Apr;18(4):1302-11. doi: 10.1091/mbc.e06-06-0515. Epub 2007 Feb 7.
Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.
X连锁凋亡抑制蛋白（XIAP）的翻译是通过内部核糖体进入位点（IRES）介导的起始过程进行的，这一过程在生理上很重要，因为在帽依赖性翻译受损的情况下，如细胞应激时，XIAP的表达对细胞存活至关重要。内部起始的调控需要IRES反式作用因子（ITAFs）与IRES元件相互作用。我们利用RNA亲和层析法鉴定XIAP的ITAFs，并分离出不均一核核糖核蛋白A1（hnRNP A1）。我们发现hnRNP A1在体外和体内均与XIAP IRES RNA相互作用，且hnRNP A1对XIAP IRES活性具有负调控作用。此外，当hnRNP A1在细胞质中积累时，XIAP IRES依赖性翻译显著降低。渗透休克是一种导致hnRNP A1在细胞质中积累的细胞应激，也会导致XIAP水平下降，而敲低hnRNP A1的表达可消除这种下降。这些结果表明，hnRNP A1的亚细胞定位是其负调控XIAP IRES活性能力的重要决定因素，提示ITAFs的亚细胞分布在调控IRES依赖性翻译中起关键作用。我们的研究结果表明，细胞质中的hnRNP A1是XIAP IRES依赖性翻译的负调控因子，揭示了这种蛋白质细胞质形式的一种新功能。
