HuR binding to cytoplasmic mRNA is perturbed by heat shock
HuR binding to cytoplasmic mRNA is perturbed by heat shock
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DOI：
10.1073/pnas.97.7.3073
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发表时间：
2000-03-28
影响因子：
11.1
通讯作者：
Steitz, JA
中科院分区：
文献类型：
--
作者：
Gallouzi, IE;Brennan, CM;Steitz, JA
位于3‘端非翻译区的富Au元件(Ares)针对编码许多原癌蛋白、细胞因子和淋巴因子的mRNAs进行快速降解。HUR是胚胎致死异常视觉(ELAV)RNA结合蛋白家族中普遍表达的成员，它结合的是序列，当在瞬时转染的细胞中过表达时，它选择性地稳定含有ARE的报告mRNAs。HUR以核质为主，但已被证明通过一种新的穿梭序列HNS在细胞核和细胞质之间穿梭。我们报道了一种免疫印迹和免疫沉淀都能识别Hur蛋白的鼠单抗3A2；它识别位于Hur的三个RNA识别基序中的第一个表位。这种抗体被用来探测热休克前后Hur与mRNA的相互作用，据报道，这种情况可以稳定含有ARE的mRNAs。在37℃时，大约三分之一的细胞质Hur出现多聚体相关，在体内UV交联表明Hur与Poly(A)(+)RNA的相互作用主要是细胞质的，而不是核的。这包括HUR在体内直接与mRNA相互作用的证据。热休克后，12-15%的HUR聚集在细胞质中的离散灶中，但令人惊讶的是，大多数HUR与核多聚(A)(+)RNA交联，其水平在应激细胞中显著增加。Hur的这一行为不同于另一种ARE结合蛋白hnRNP D的行为，hnRNP D被认为是mRNA衰退而不是mRNA稳定的效应器，以及一般的前RNA结合蛋白hnRNP A1。我们将这些差异解释为意味着HUR与含ARE的mRNAs的时间关联不同于其他两种蛋白质。
AU-rich elements (AREs) located in the 3' untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal Vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)(+) RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12-15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)(+) RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.