Nuclear import and retention domains in the amino terminus of RECQL4.
Mutations in a human RecQ helicase homologue, RECQL4, have been identified in patients with Type II Rothmund-Thomson syndrome (RTS) with osteosarcoma predisposition, RAPADILINO syndrome, and Baller-Gerold syndrome. A role in DNA replication initiation has been demonstrated and mapped to the amino terminus upstream of the helicase domain; however, no nuclear localization signal (NLS) has been identified by sequence analysis. Here, we show both endogenous and green fluorescent protein (GFP)-tagged RECQL4 are nuclear and cytoplasmic in transformed cell lines. Using GFP-tagged constructs we identified a major nuclear localization domain within amino acids (aa) 363-492 (exons 5-8) sufficient for nuclear localization of GFP and necessary for nuclear localization of RECQL4 as GFP-RECQL4 deleted for aa 363-492 is entirely cytoplasmic. Additional mapping within this domain revealed that a conserved block of 22 basic amino acids (aa 365-386; exons 5-6) is sufficient for nuclear localization of GFP, but not required for nuclear import of RECQL4. Conversely, even though the region encoded by exon 7-8 is not sufficient for nuclear import of GFP, GFP-RECQL4 deleted for exon 7 (aa 420-463), a mutation found in all reported patients with RAPADILINO syndrome, is cytoplasmic. Nuclear localization of the exon 7 deletion construct is increased in cells treated with leptomycin B suggesting that exon 7 encodes a domain required for nuclear retention of RECQL4. This retention activity is partially conveyed by a conserved VLPLY motif (aa 450-454) in exon 7 of the human sequence. In summary, unlike other RecQ proteins with carboxyl terminal NLS, RECQL4 nuclear localization and retention activities are amino terminal. This location would provide nuclear transport of putative truncated proteins encoded by RTS mutant alleles consistent with the proposed essential replication function in the amino terminus of RECQL4.
Burks
Lynnette M
LM
Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, TX 77030, USA.
Yin
Jinhu
J
Plon
Sharon E
SE
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics; D053484:RecQ Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D005231:Fatty Acids, Unsaturated; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C506731:RECQL4 protein, human; D053484:RecQ Helicases; C038753:leptomycin B