Subcellular Distribution of the Human Putative Nucleolar GTPase GNL1 is Regulated by a Novel Arginine/Lysine-Rich Domain and a GTP Binding Domain in a Cell Cycle-Dependent Manner

Laboratory of Molecular Virology and Cell Biology, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India.
Journal of Molecular Biology (Impact Factor: 4.33). 02/2012; 416(3):346-66. DOI: 10.1016/j.jmb.2011.12.066
Source: PubMed


GNL1, a putative nucleolar GTPase, belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. Members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization with respect to cell growth remain unknown. To understand the nuclear transport mechanism of GNL1, we have identified a novel arginine/lysine-rich nucleolar localization signal in the NH(2)-terminus that is shown to translocate GNL1 and a heterologous protein to the nucleus/nucleolus in a pathway that is independent of importin-α and importin-β. In addition, the present investigation provided evidence that GNL1 localized to the nucleus and the nucleolus only in G2 stage, in contrast to its cytoplasmic localization in the G1 and S phases of the cell cycle. Using heterokaryon assay, we have demonstrated that GNL1 shuttles between the nucleus and the cytoplasm and that the motif between amino acids 201 and 225 is essential for its export from the nucleus by a signal-mediated CRM1-independent pathway. Alanine-scanning mutagenesis of conserved residues within G-domains suggests that the G2 motif is critical for guanine nucleotide triphosphate (GTP) binding of GNL1 and further showed that nucleolar retention of GNL1 is regulated by a GTP-gating-mediated mechanism. Expression of wild-type GNL1 promotes G2/M transition, in contrast to the G-domain mutant (G2m), which fails to localize to the nucleolus. These data suggest that nucleolar translocation during G2 phase may be critical for faster M-phase transition during cell proliferation. Replacement of conserved residues within the G5 motif alters the stability of GNL1 without changing GTP binding activity. Finally, our data suggest that ongoing transcription is essential for the efficient localization of GNL1 to the nucleolus. Overall, the results reported here demonstrate that multiple mechanisms are involved in the translocation of GNL1 to the nucleolus in a cell cycle-dependent manner to regulate cell growth and proliferation.


Available from: Suryaraja R
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell autonomous and non-cell autonomous growth control roles of NS3 in Drosophila, and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.
    Genetics 02/2013; 194(1). DOI:10.1534/genetics.112.149104 · 5.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The contribution of the nucleolus to cancer is well established with respect to its traditional role in facilitating ribosome biogenesis and proliferative capacity. More contemporary studies however, infer that nucleoli contribute a much broader role in malignant transformation. Specifically, extra-ribosomal functions of the nucleolus position it as a central integrator of cellular proliferation and stress signaling, and are emerging as important mechanisms for modulating how oncogenes and tumour suppressors operate in normal and malignant cells. The dependence of certain tumour cells to co-opt nucleolar processes to maintain their cancer phenotypes has now clearly been demonstrated by the application of small molecule inhibitors of RNA Polymerase I to block ribosomal DNA transcription and disrupt nucleolar function (Bywater et al., 2012 [1]). These drugs, which selectively kill tumour cells in vivo while sparing normal cells, have now progressed to clinical trials. It is likely that we have only just begun scratch the surface of the potential of the nucleolus as a new target for cancer therapy, with "suppression of nucleolar stress" representing an emerging "hallmark" of cancer.
    Biochimica et Biophysica Acta 01/2014; 1842(6). DOI:10.1016/j.bbadis.2013.12.009 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objective: To investigate whether a conserved HLA class I region influenced the development of Graves' ophthalmopathy (GO) in patients with Graves' disease (GD) in a Taiwan-Chinese population. Design: Case-control study. Participants: Four hundred sixty-eight Taiwan-Chinese patients with GD; 200 of these patients had GO, whereas 268 patients did not. Methods: Five single nucleotide polymorphisms (SNPs) between the HLA-A and HLA-C loci were genotyped. Main Outcome Measures: The Mann-Whitney U test and chi-square test with Bonferroni correction were used. The odds ratios (ORs) were estimated by applying unconditional logistic regression with a 95% confidence intervals (CIs). Results: Strong gender effects on the distribution of the SNPs were apparent: male GD patients carrying an A allele at rs2074503 in the PRR3 gene tended to avoid demonstrating GO (P = 0.008; OR, 0.450; 95% CI, 0.248-0.819), whereas female patients tended to show GO (P = 0.01; OR, 1.486; 95% CI, 1.098-2.012). In addition, only the female GD patients with a T allele at rs1264439 in the ABCF-1 gene tended to demonstrate GO (P = 0.005; OR, 1.539; 95% CI, 1.139-2.081). Analysis of the haplotype blocks of the SNPs rs2074505 (GNL1) and rs2074503 (PRR3) showed that haplotype HA1 was underrepresented in male GO patients (P = 0.004; OR, 0.418; 95% CI, 0.228-0.767), whereas HA-4 was underrepresented in female GO patients (P = 0.007; OR, 0.660; 95% CI, 0.490-0.895). Conclusions: The results suggested that SNPs at PRR3 and ABCF1 genes and the haplotype composed by SNPs at GNL1 and PRR3 between the HLA-A and HLA-C genes tended to predict GO in a gender-dependent manner in patients with GD in Taiwan. (C) 2014 by the American Academy of Ophthalmology.
    Ophthalmology 06/2014; 121(10). DOI:10.1016/j.ophtha.2014.04.027 · 6.14 Impact Factor
Show more