Sanford Jeremy R, Ellis Jonathan D, Cazalla Demian, Cáceres Javier F
Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom.
Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15042-7. doi: 10.1073/pnas.0507827102. Epub 2005 Oct 6.
The Ser/Arg-rich (SR) proteins constitute a family of highly conserved nuclear phosphoproteins that are involved in many steps of mRNA metabolism. Previously, we demonstrated that shuttling SR proteins can associate with translating ribosomes and enhance translation of reporter mRNAs both in vivo and in vitro. Here, we show that endogenous, cytoplasmic splicing factor 2/alternative splicing factor (SF2/ASF) associated with the translation machinery is hypophosphorylated, suggesting that the phosphorylation state of the Arg-Ser-rich (RS) domain may influence the role of SF2/ASF in cytoplasmic RNA processing. In agreement, we show that mutations mimicking a hypophosphorylated RS domain strongly increased SF2/ASF binding to cytoplasmic mRNA and its activity in translation. We also demonstrate that, whereas the RS domain is not required for the function of SF2/ASF in mRNA translation in vivo or in vitro, its second RNA recognition motif (RRM)2 plays a critical role in this process. Taken together, these data suggest that RS-domain phosphorylation may influence the association of SF2/ASF with mRNA, whereas RRM2 may play an important role in mediating protein-protein interactions during translation. These data are consistent with a model whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins.
富含丝氨酸/精氨酸（SR）的蛋白质构成了一个高度保守的核磷蛋白家族，它们参与mRNA代谢的多个步骤。此前，我们证明穿梭SR蛋白可与正在翻译的核糖体结合，并在体内和体外增强报告基因mRNA的翻译。在此，我们表明与翻译机制相关的内源性细胞质剪接因子2/可变剪接因子（SF2/ASF）处于低磷酸化状态，这表明富含精氨酸-丝氨酸（RS）结构域的磷酸化状态可能影响SF2/ASF在细胞质RNA加工中的作用。与此一致的是，我们发现模拟低磷酸化RS结构域的突变会强烈增加SF2/ASF与细胞质mRNA的结合及其在翻译中的活性。我们还证明，虽然RS结构域在体内或体外mRNA翻译中对SF2/ASF的功能不是必需的，但其第二个RNA识别基序（RRM）2在此过程中起关键作用。综上所述，这些数据表明RS结构域的磷酸化可能影响SF2/ASF与mRNA的结合，而RRM2可能在翻译过程中介导蛋白质-蛋白质相互作用中发挥重要作用。这些数据与一种模型一致，即可逆的蛋白质磷酸化差异调节穿梭SR蛋白的亚细胞定位和活性。
