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First published online 10 June 2003
doi: 10.1242/jcs.00618

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DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly

Mónica Álvarez1, Xavier Estivill1 and Susana de la Luna1,2,*

1 Program in Genes and Disease, Centre de Regulació Genòmica-CRG, Passeig Marítim 37-49, 08003-Barcelona, Spain
2 Institut per la Recerca i Estudis Avançats-ICREA, Barcelona, Spain



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  		Fig. 1. Subcellular localization of DYRK1A. (A) Schematic representation of a series of HA-tagged deletion mutants covering the full-length open reading frame of human DYRK1A (754 amino acids alternative spliced form). Representative domains are indicated: NLS (nuclear localization signal); KINASE (kinase catalytic domain); PEST (domain rich in proline, glutamate, serine and threonine); His (histidine repeat) and Ser/Thr (serine/threonine-rich region). {Delta}, deletion; N, nuclear accumulation; SP, speckle-like staining pattern. All these constructs were transiently transfected in COS-7 cells. (B) Western blot of total protein extracts (10% SDS-PAGE) using an anti-HA antibody for protein detection. Positions of marker proteins (in kDa) are indicated. (C) Indirect immunofluorescence analysis using an anti-HA monoclonal antibody, followed by an FITC-conjugated rabbit anti-mouse secondary antibody. Results for the most representative mutants are shown in B and C.

 


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  		Fig. 2. Identification of a novel NLS in the catalytic domain of DYRK1A. The GFP-DYRK1A fusions indicated were expressed by transient transfection in COS-7 cells, and their subcellular localization was analyzed by fluorescence microscopy. The sequence and position of the second NLS motif predicted and the polybasic sequence element adjacent to it are indicated. The DYRK1A amino-acid residues of the different GFP fusions are also indicated.

 


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  		Fig. 3. Accumulation of DYRK1A in speckle-like compartments is mediated by the histidine domain. Schematically depicted GFP-DYRK1A fusion proteins, containing a region including the NLS2 (378-588), the histidine repeat (590-616) or both (378-616). The amino-acid sequence of the DYRK1A histidine repeat is indicated. Fluorescence images of COS-7 transfected cells are shown.

 


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  		Fig. 4. DYRK1A colocalizes with splicing factors in nuclear speckles. COS-7 cells were transfected with GFP-DYRK1A wildtype (A-C), GFP-DYRK1A 590-616 (D), and GFP-cyclin T1 433-533 and 503-533 (E) and immunostained with reported markers of different subnuclear compartments. In A, POD nuclear bodies were detected with an anti-PML antibody. In B, cells were cotransfected with HA-SUMO, and SUMO-conjugated dots were detected by using an anti-HA antibody. In C-E, SFCs were detected either with an antibody to the SR splicing factor SC35 (C, upper panel, D and E) or with an anti-Sm antibody (Y12) that recognizes sRNPs (C, lower panel). GFP fusion proteins were visualized directly by fluorescence microscopy (left column, green) and nuclear subcompartments by indirect immunofluorescence, using Cy3-conjugated goat anti-rabbit (A) and Texas Red-labeled sheep anti-mouse (B-E) as secondary antibodies (middle column, red). Merged images are also shown (right column). All the images in this figure, except in E, were taken by confocal microscopy for clearer confirmation of the colocalization results.

 


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  		Fig. 5. DYRK1A accumulation in nuclear speckles is not dependent on its kinase activity. (A) COS-7 cells were transfected with wild-type HA-DYRK1A, the mutant HA-DYRK1A K179R and the empty vector as a control. HA-tagged proteins were immunoprecipitated with an anti-HA antibody, and the immunocomplexes subjected to an in vitro kinase assay using the synthetic peptide DYRKtide as an exogenous substrate. A western blot for both inputs (i) and immunoprecipitates (IP) was developed with an anti-HA antibody to control for equal presence of the fusion proteins (left panel). Autophosphorylation of DYRK1A was detected by resolving the kinase reactions in 10% SDS-PAGE and autoradiography of the dried gels (middle panel). Incorporation of 32P into DYRKtide was determined in triplicate by dotting aliquots of the reaction onto phosphocellulose paper. The average count is shown on the chart (right panel). (B) COS-7 cells were transfected with GFP-fusion proteins of DYRK1A wt (upper panel) and the kinase-negative mutant described above (DYRK1A K179R) (lower panel) and co-stained with the antibody anti-SC35 to detect the colocalization of DYRK1A and SC35 in nuclear speckles.

 


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  		Fig. 6. DYRK1A localization responds to the transcriptional state of the cell. COS-7 cells were transfected with GFP-fusion proteins of DYRK1A wildtype (A), the catalytically inactive mutant (B) and the histidine domain (C) and treated with {alpha}-amanitin (50 µg/ml, 5 hours) to inhibit transcription. Speckles labeled with anti-SC35 became larger, rounded-up and overlapped with DYRK1A.

 


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  		Fig. 7. The effect of DYRK1A overexpression on the distribution of SC35 splicing factor is dependent on DYRK1A kinase activity. (A) A fluorescent image of a cell overexpressing GFP-DYRK1A, where nuclear speckles for both DYRK1A (green) and endogenous SC35 (red) appeared diffuse, and an untransfected cell showing the normal distribution of SC35. The nuclei were counterstained with DAPI (right). The GFP-fusion proteins of DYRK1A (B), as well as DYRK1B and a chimeric protein, DYRK1B/DYRK1A, with the C-terminal regions exchanged (C) were transfected and cells immunostained with anti-SC35. The percentage of transfected cells, which presented accumulation in speckles for the endogenous SC35 splicing factor, is represented in the charts to establish the extent of speckle disassembly induced by DYRK1A overexpression. The data are the average±s.d. of two independent experiments with at least 250 cells counted in each. Images of cells overexpressing DYRK1B, and the chimeric DYRK1B/A, co-stained with SC35, are also shown (C, right panel).

 

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© The Company of Biologists Ltd 2003