Suzuki Maiko, Iijima Megumi, Nishimura Akira, Tomozoe Yusuke, Kamei Daisuke, Yamada Michiyuki
Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.
FEBS J. 2005 Aug;272(15):3975-87. doi: 10.1111/j.1742-4658.2005.04820.x.
Heterogeneous nuclear ribonucleoprotein (hnRNP) D/AUF1 functions in mRNA genesis in the nucleus and modulates mRNA decay in the cytoplasm. Although it is primarily nuclear, it shuttles between the nucleus and cytoplasm. We studied the nuclear import and export of the last exon-encoding sequence common to all its isoforms by its expression as a green fluorescent protein-fusion protein in HeLa cells and by heterokaryon assay. The C-terminal 19-residue sequence (SGYGKVSRRGGHQNSYKPY) was identified as an hnRNP D nucleocytoplasmic shuttling sequence (DNS). In vitro nuclear transport using permeabilized cells indicated that nuclear import of DNS is mediated by transportin-1 (Trn-1). DNS accumulation in the nucleus was dependent on Trn-1, Ran, and energy in multiple rounds of nuclear transport. Use of DNS with deletions, alanine scanning mutagenesis and point mutations revealed that two separate regions (the N-terminal seven residues and the C-terminal two residues) are crucial for in vivo and in vitro transport as well as for interaction with Trn-1. The N- and C-terminal motifs are conserved in the shuttling sequences of hnRNP A1 and JKTBP.
不均一核核糖核蛋白（hnRNP）D/AUF1在细胞核中的mRNA生成过程中发挥作用，并调节细胞质中的mRNA降解。尽管它主要位于细胞核中，但会在细胞核和细胞质之间穿梭。我们通过在HeLa细胞中表达绿色荧光蛋白融合蛋白并进行异核体分析，研究了其所有亚型共有的最后一个外显子编码序列的核输入和输出。C末端的19个氨基酸序列（SGYGKVSRRGGHQNSYKPY）被鉴定为hnRNP D核质穿梭序列（DNS）。使用通透细胞进行的体外核运输表明，DNS的核输入由运输蛋白-1（Trn-1）介导。在多轮核运输中，DNS在细胞核中的积累依赖于Trn-1、Ran和能量。使用缺失、丙氨酸扫描诱变和点突变的DNS表明，两个独立的区域（N末端的七个氨基酸和C末端的两个氨基酸）对于体内和体外运输以及与Trn-1的相互作用至关重要。N末端和C末端基序在hnRNP A1和JKTBP的穿梭序列中保守。
