Gallouzi I E, Brennan C M, Steitz J A
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
RNA. 2001 Sep;7(9):1348-61. doi: 10.1017/s1355838201016089.
AU-rich elements (AREs) located in the 3' UTRs of the messenger RNAs (mRNAs) of many mammalian early response genes promote rapid mRNA turnover. HuR, an RRM-containing RNA-binding protein, specifically interacts with AREs, stabilizing these mRNAs. HuR is primarily nucleoplasmic, but shuttles between the nucleus and the cytoplasm via a domain called HNS located between RRM2 and RRM3. We recently showed that HuR interacts with two protein ligands, pp32 and APRIL, which are also shuttling proteins, but rely on NES domains recognized by CRM1 for export. Here we show that heat shock induces increased association of HuR with pp32 and APRIL through protein-protein interactions and that these ligands partially colocalize with HuR in cytoplasmic foci. HuR associations with the hnRNP complex also increase, but through RNA links. CRM1 coimmunoprecipitates with HuR only after heat shock, and nuclear export of HuR becomes sensitive to leptomycin B, an inhibitor of CRM1. Export after heat shock requires the same domains of HuR (HNS and RRM3) that are essential for binding pp32 and APRIL. In situ hybridization and coimmunoprecipitation experiments show that LMB treatment blocks both hsp70 mRNA nuclear export and its cytoplasmic interaction with HuR after heat shock. Together, our results argue that upon heat shock, HuR switches its export pathway to that of its ligands pp32 and APRIL, which involves the nuclear export factor CRM1. HuR and its ligands may be instrumental in the nuclear export of heat-shock mRNAs.
富含AU元件（AREs）位于许多哺乳动物早期反应基因信使核糖核酸（mRNAs）的3'非翻译区（UTRs），可促进mRNA的快速周转。HuR是一种含RNA识别基序（RRM）的RNA结合蛋白，它与AREs特异性相互作用，使这些mRNA稳定。HuR主要位于核质中，但通过位于RRM2和RRM3之间的一个称为HNS的结构域在细胞核和细胞质之间穿梭。我们最近发现，HuR与两种蛋白质配体pp32和增殖诱导配体（APRIL）相互作用，这两种配体也是穿梭蛋白，但依赖于被CRM1识别的核输出信号（NES）结构域进行输出。在这里我们表明，热休克通过蛋白质-蛋白质相互作用诱导HuR与pp32和APRIL的结合增加，并且这些配体在细胞质病灶中与HuR部分共定位。HuR与不均一核糖核蛋白（hnRNP）复合物的结合也增加，但通过RNA连接。仅在热休克后，CRM1与HuR共免疫沉淀，并且HuR的核输出对CRM1的抑制剂雷帕霉素B敏感。热休克后的输出需要HuR的相同结构域（HNS和RRM3），这些结构域对于结合pp32和APRIL至关重要。原位杂交和共免疫沉淀实验表明，雷帕霉素B处理在热休克后阻断了热休克蛋白70（hsp70）mRNA的核输出及其与HuR的细胞质相互作用。总之，我们的结果表明，热休克时，HuR将其输出途径切换为其配体pp32和APRIL的输出途径，这涉及核输出因子CRM1。HuR及其配体可能在热休克mRNA的核输出中起作用。
