Nuclear import of the U1A splicesome protein is mediated by importin alpha /beta and Ran in living mammalian cells.
U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100-144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin alpha/beta and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin alpha. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin alpha/beta and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin beta binding domain of importin alpha. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin alpha/beta and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.
Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka and Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.
M
M
Hieda
T
T
Tachibana
M
M
Fukumoto
Y
Y
Yoneda
D000595:Amino Acid Sequence; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002474:Cell-Free System; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D028884:Karyopherins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D049452:Green Fluorescent Proteins; D020931:ran GTP-Binding Protein