Krebber H, Taura T, Lee M S, Silver P A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and The Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Genes Dev. 1999 Aug 1;13(15):1994-2004. doi: 10.1101/gad.13.15.1994.
Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.
Npl3p是酿酒酵母中主要的mRNA结合蛋白，穿梭于细胞核和细胞质之间。Npl3p羧基末端的单个氨基酸变化（E409→K）使突变蛋白主要定位于细胞质中，因为其导入细胞核的过程出现延迟。增加Npl3p的核输入受体Mtr10p的细胞内浓度，可以逆转这种输入缺陷。相反，利用这个突变体，我们发现，在编码含NES蛋白的输出受体的XPO1/CRM1以及编码一种必需核孔蛋白的RAT7发生突变的细胞中，Npl3p和mRNA从细胞核输出的过程显著减慢。有趣的是，在热休克、高盐或乙醇诱导应激后，即在大多数mRNA输出受阻的情况下，Npl3p仍能从细胞核输出。应激诱导的Npl3p输出既不依赖于Xpo1p的活性，也不依赖于应激后发生的热休克mRNA的持续选择性输出。紫外线交联实验表明，在正常条件下Npl3p与mRNA结合，但在应激细胞中不再与RNA相关联。综上所述，我们认为Npl3p以及可能其他的mRNA结合蛋白在细胞核中与mRNA解偶联，提供了一个调节mRNA输出的通用开关。根据这个模型，在正常条件下，Npl3p是有输出能力的核糖核蛋白复合体的主要成分。然而，在应激条件下，Npl3p不再与输出复合体结合，使其失去输出能力，从而滞留在细胞核中。
