Identification of the nucleocytoplasmic shuttling sequence of heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its interaction with mRNA.
JKTBP proteins are related to a family of heterogeneous nuclear ribonucleoproteins (hnRNPs) that function in mRNA biogenesis and mRNA metabolism. JKTBP proteins constituted of isoforms 1, 2, and 1Delta6 are localized in the nucleus. We show that the dominant form JKTBP1 shuttles between the nucleus and the cytoplasm and interacts with mRNA. Immunofluorescence microscopy and immunoblotting of the subcellular fractions and overexpression of JKTBP tagged with green fluorescent protein indicated that JKTBP1 and JKTBP1Delta6, but not JKTBP2, accumulate in the cytoplasm upon polymerase II transcription inhibition. After release from inhibition, the return of accumulated cytoplasmic JKTBP to the nucleus was temperature-dependent. In heterokaryons, green fluorescent protein-tagged JKTBP1 and JKTBP1Delta6 migrated from the HeLa nucleus to the mouse nucleus, but JKTBP2 did not. Using various JKTBP deletion mutants, the 25-residue C-terminal tail was identified as a shuttling sequence like M9. It is conserved in the C-terminal tails of hnRNP D/AUF1 and type A/B hnRNP/ABBP-1. Analysis of its sequence-specific interacting protein indicated that JKTBP nuclear import is mediated by the receptor transportin 1/karyopherin beta2. UV cross-linking revealed the increased occurrence of JKTBP1 directly interacting with poly(A)(+) RNA in the cytoplasm following actinomycin D treatment. We discuss a role of JKTBP in mRNA nuclear export.
Kawamura
Hidenobu
H
Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan.
Tomozoe
Yusuke
Y
Akagi
Tadayuki
T
Kamei
Daisuke
D
Ochiai
Michiru
M
Yamada
Michiyuki
M
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D015151:Immunoblotting; D028884:Karyopherins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011061:Poly A; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013696:Temperature; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; C111986:HNRPDL protein, human; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C484734:Hnrpdl protein, mouse; D028884:Karyopherins; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; D011061:Poly A; D005982:Glutathione Transferase