Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence.
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.
Suzuki
Maiko
M
Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.
Iijima
Megumi
M
Nishimura
Akira
A
Tomozoe
Yusuke
Y
Kamei
Daisuke
D
Yamada
Michiyuki
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006367:HeLa Cells; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology
D000596:Amino Acids; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D020033:Protein Isoforms; D021382:Protein Sorting Signals