Nuclear import of the stem-loop binding protein and localization during the cell cycle.
A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem-loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impalpha/Impbeta and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impalpha/Impbeta binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impalpha/Impbeta pathway contributes to SLBP nuclear import in HeLa cells.
Erkmann
Judith A
JA
Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Wagner
Eric J
EJ
Dong
Jian
J
Zhang
Yanping
Y
Kutay
Ulrike
U
Marzluff
William F
WF
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015153:Blotting, Western; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D016195:G2 Phase; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D017386:Sequence Homology, Amino Acid; D028901:alpha Karyopherins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006657:Histones; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C092663:SLBP protein, human; D028901:alpha Karyopherins; D028961:beta Karyopherins; D039221:mRNA Cleavage and Polyadenylation Factors; C424165:transportin SR2; D049452:Green Fluorescent Proteins; D000409:Alanine