Determination of the functional domains involved in nucleolar targeting of nucleolin.
Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.
Créancier
L
L
Laboratoire de Biologie Moleculaire Eucaryote, Institut de Biologie Cellulaire et Genetique, Centre National de la Recherche Scientifique, Toulouse, France.
Prats
H
H
Zanibellato
C
C
Amalric
F
F
Bugler
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D000097796:Nucleolin
D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000097796:Nucleolin