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First published online 8 November 2005
doi: 10.1242/jcs.02669

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Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

Tong Zhang, Nathalie Delestienne, Georges Huez, Véronique Kruys^*, and Cyril Gueydan^*

Laboratoire de Chimie Biologique, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, 6041 Gosselies, Belgium

View larger version (69K): [in a new window]   Fig. 1. TIAR accumulates in the cytoplasm of cells treated with actinomycin D. Cos cells grown on glass coverslips were incubated for 3 hours in normal medium (NT) or in medium containing cycloheximide (CHX; 20 µg/ml) or actinomycin D (ActD; 5 µg/ml) or both agents. Cells were then fixed, immunostained with anti-TIAR antibody and analyzed by confocal fluorescence microscopy as described in the Materials and Methods. Phase-contrast transmission images are also shown (Phase). Translational blockade by cycloheximide was verified by [35S]methionine incorporation (data not shown).

View larger version (24K): [in a new window]   Fig. 2. Subcellular localization of EGFP-TIAR hybrid proteins. (A) Schematic representation of TIAR protein and TIAR mutants fused to EGFP protein. The amino acids bordering the different domains composing TIAR are indicated. The dotted lines indicate the deleted region in the different mutants. (B) Subcellular distribution of GFP and GFP-TIAR hybrid proteins (represented in A) were analyzed by epifluorescence microscopy. Cell nuclei were stained with DAPI. (C) Ratios of cytoplasmic (C) and nuclear (N) fluorescence observed for GFP alone or fused with TIAR or relevant domains of TIAR. These ratios are the means±s.d. and were calculated as described in the Materials and Methods.

View larger version (19K): [in a new window]   Fig. 3. Subcellular localization of EGFP-TIAR hybrid proteins lacking parts of the first half of the auxiliary domain. (A) Schematic representation of TIAR protein and TIAR mutants fused to EGFP. The amino acid sequence of the first half of the auxiliary domain as well as the deletions introduced in the different mutants is specified. (B) The subcellular distribution of GFP and GFP-TIAR hybrid proteins was analyzed by epifluorescence microscopy and quantified as for Fig. 2C.

View larger version (28K): [in a new window]   Fig. 4. RRM3 mediates TIAR nuclear export. (A) Subcellular localization of TIAR-FLAG and TIARRRM3-FLAG mutant in Cos cells before and after actinomycin D (5 µg/ml) treatment for 3 hours. Cells were fixed, immunostained with anti-FLAG antibody, and analyzed by epifluorescence microscopy. (B) Schematic representation of NPc-NLS-FLAG constructs with or without different parts of the TIAR protein. Mutations in the NPc-RRM3mutRNP2-NLS-FLAG and NPc-RRM3mutRNP1-NLS-FLAG were: Y208D, C209R, G210P, G211P, I212D and G241P, Y242D, F244D, V245E, F247D, respectively. (C) Subcellular localization of proteins illustrated in B.

View larger version (17K): [in a new window]   Fig. 5. Characterization of the nucleo-cytoplasmic distribution of TIAR and TIAR mutants by cell fractionation. Cos-7 cells were transfected with the indicated TIAR and TIAR mutants fused with the FLAG epitope. Where indicated, cells were then treated with actinomycin D (5 µg/ml, 3 hours) before cell fractionation. Cytoplasmic (C) and nuclear (N) extracts were analyzed by western blot using anti-FLAG and anti-FBP to control the fractionation procedure.

View larger version (74K): [in a new window]   Fig. 6. TIAR nuclear export is independent of CRM1 export pathway. (A) Cos cells grown on glass coverslips were incubated for 3 hours in normal medium (NT) or in medium containing actinomycin D (ActD; 5 µg/ml) or leptmomycin B (LMB; 0.2 µM) or both agents. Cells were then fixed, immunostained with anti-TIAR antibody and analyzed by confocal fluorescence microscopy as described in the Materials and Methods. Phase-contrast images are also shown (Phase). (B) Cos cells grown on coverslips were transfected with the GFP-MK2-T205,317E construct. Thirty-six hours after transfection, cells were incubated or not with leptomycin B and GFP fluorescence was analyzed as in A.

View larger version (45K): [in a new window]   Fig. 7. ATP depletion leads to the cytoplasmic accumulation of hnRNP A1 and TIAR. Cos cells grown on coverslips were ATP-depleted and immunostained. The subcellular localization of hnRNP A1 and TIAR was analyzed as described in Fig. 1.

View larger version (19K): [in a new window]   Fig. 8. TIAR and TIA-1 subcellular localization relies on the same sequence determinants. (A) Schematic representation of the TIA-1 constructs. The amino acids bordering the different TIA-1 domains are indicated. The dotted lines indicate the deleted region in the different mutants. (B,C) Subcellular distribution of GFP-TIA-1, GFP-TIA-1 mutants and NPc-RRM3(TIA-1)-NLS-FLAG described in A. The experiment was performed as described in Fig. 2B and Fig. 4C and ratios of cytoplasmic and nuclear fluorescence were calculated as previously described.