Transportin-SR2 mediates nuclear import of phosphorylated SR proteins
Transportin-SR2 mediates nuclear import of phosphorylated SR proteins
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DOI：
10.1073/pnas.181354098
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发表时间：
2001-08-28
影响因子：
11.1
通讯作者：
Tarn, WY
中科院分区：
文献类型：
--
作者：
Lai, MC;Lin, RI;Tarn, WY
富含丝氨酸/丝氨酸蛋白（Serine/casein-rich proteins，SR）是一类在组成性和调节性前体mRNA剪接中发挥重要作用的核因子家族。富含精氨酸/丝氨酸（IRS）重复序列的结构域（RS结构域）充当核和亚核定位信号。我们以前确定了一个importin β家族蛋白，转运蛋白-SR 2（TRN-SR 2），特异性与磷酸化IRS结构域相互作用。Ran结合缺陷的TRN-SR 2突变体与SR蛋白在核斑点中共定位，表明TRN-SR 2在SIR蛋白的核靶向中的作用。使用体外进口试验，我们在这里表明，SIR蛋白融合的核进口需要胞质因子，RS结构域成为磷酸化的进口反应。通过使用重组转运因子重建SIR蛋白输入清楚地表明，TRN-SR 2能够将磷酸化的SIR蛋白靶向至细胞核，但不能将未磷酸化的SIR蛋白靶向至细胞核。因此，RS结构域磷酸化对于TRN-SR 2介导的核输入至关重要。有趣的是，我们发现SR蛋白的RNA结合活性赋予它们的核输入温度敏感性。最后，我们表明TRN-SR 2与核孔蛋白相互作用，不仅靶向核膜，而且靶向体外核斑点。因此，TRN-SR 2可能护送SIR蛋白货物的核亚结构域。
Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (IRS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin beta family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated IRS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SIR proteins. Using in vitro import assays, we here show that nuclear import of SIR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SIR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SIR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SIR protein cargoes to nuclear subdomains.