Journal: RNA Biology
Article Title: DUSP11 – An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells
doi: 10.1080/15476286.2017.1306169
Figure Lengend Snippet: DUSP11 motifs/domains involved in localization and RNA phosphatase activity in cells. (A) Schematic diagram of DUSP11 (not to scale) illustrating the relative location of the arginine-rich motifs, putative NLS, catalytic domain, and proline-rich motif. Below are the DUSP11 mutants that we generated, which are described in the text. We cloned the constructs into pcDNA3.1+ and tagged the N-terminus via PCR-based engineering with a 3xFLAG epitope. (B) Nuclear/cytosolic fractionation of A549 and HEK293T cells to determine localization of DUSP11. Cells from one well of 12-well format plate were trypsinized, washed with phosphate-buffered solution (PBS), resuspended in 50-ul of CSKT buffer [10mM PIPES (pH 6.8), 100mM NaCl, 300mM sucrose, 3mM MgCl 2 , 1mM EDTA, 1mM DTT, 0.5% (vol/vol) tritonX-100, protease inhibitors (Roche)], and placed on ice for 10 minutes. Nuclei were then pelleted by centrifugation at 5000xg for 5 minutes. The supernatant [cytosolic fraction (Cyto)] was removed (50-ul) and added to 50-ul of SDS lysis buffer (1% SDS; 2% 2-mercaptoethanol). The nuclei were then washed in 50-ul of CSKT buffer and centrifuged at 5000xg for 5 minutes. The nuclei were then resuspended in 50-ul of water followed by addition of 50-ul of SDS buffer to make the nuclear fraction (Nuc). A whole cell lysate (WCL) was collected in parallel by trypsinization and washing of the cells, which were then resuspended in 50-ul of water, followed by addition of 50-ul of SDS lysis buffer. Samples were boiled for 10 minutes followed by vortexing for 30 seconds. Equal volumes (10-ul) of fractions were then fractionated via 12.5% SDS-PAGE. Protein was then transferred to nitrocellulose membrane (Bio-Rad). Anti-DUSP11 polyclonal rabbit antibody (1:2000 dilution; Proteintech, catalog no. 10204–2-AP), anti-α-Tubulin monoclonal mouse antibody (1:10,000 dilution; Sigma-Aldrich, catalog no. T6199), and anti-Histone H3 (D1H2) rabbit antibody (Cell signaling; mAb #4499) in phosphate-buffered saline (PBS) with 0.1% Tween20 (PBST) and 5% BSA were used to blot for DUSP11, α-Tubulin (cytosolic localized control), and Histone H3 (nuclear localized control). After washing with PBST, membranes were blotted with IRDye 800CW and IRDye 680LT secondary antibodies (1:10,000 dilution; LI-COR) in PBST with 5% BSA. Blots were washed 4 times with PBST and then scanned on an Odyssey CLx infrared imaging system (LI-COR). (C) Nuclear/cytosolic fractionation as described in (B) of HEK293T cells (12-well format) transfected with 200-ng/well of the 3xFLAG-tagged DUSP11 constructs from (A). The anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich) was used to stain for the 3xFLAG-tagged DUSP11 proteins. (D) Analysis of the BLV-B2 5p:3p miRNA ratios with co-expression of the DUSP11 constructs from (A). DUSP11-null HEK293T cells (24-well format; 70% confluent) were co-transfected with 5-ng/well of the EBER1 transfection control expression vector (pEBV RIJ), 400-ng/well of pBLV-B2, and 100-ng/well of pDUSP11, pDUSP11-C152, or the DUSP11 construct shown in (A). Northern blot analysis was then performed as described in refs. and . The membrane was probed with the BLV-miR-B2–5p probe, stripped and re-probed with the BLV-miR-B2–3p probe, and stripped and re-probed with the EBER1 probe as a loading/transfection control. Below is immunoblot analysis that was performed on duplicate lysates to confirm and quantitate DUSP11 expression from each construct using the anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich). (E) Bar graph of the relative band density from 4 independent RNA gel blot analyses, as shown in D (except for DUSP11-Δ13–26, in which 3 replicates were performed). Bars represent the average band density ratio (5p/3p) +/− SD of the BLV-B2 miRNAs. The ratio with co-expression of each DUSP11 construct was normalized to the C152S catalytic mutant. Asterisks represent statistical significance (p<0.05) in comparison to wild type DUSP11 (WT). P-values were determined using student's t-test.
Article Snippet: After washing with PBST, membranes were blotted with IRDye 800CW and IRDye 680LT secondary antibodies (1:10,000 dilution; LI-COR) in PBST with 5% BSA.
Techniques: Activity Assay, Generated, Clone Assay, Construct, Fractionation, Centrifugation, Lysis, SDS Page, Imaging, Transfection, Staining, Expressing, Plasmid Preparation, Northern Blot, Western Blot, Mutagenesis
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![DUSP11 motifs/domains involved in localization and RNA phosphatase activity in cells. (A) Schematic diagram of DUSP11 (not to scale) illustrating the relative location of the arginine-rich motifs, putative NLS, catalytic domain, and proline-rich motif. Below are the DUSP11 mutants that we generated, which are described in the text. We cloned the constructs into pcDNA3.1+ and tagged the N-terminus via PCR-based engineering with a 3xFLAG epitope. (B) Nuclear/cytosolic fractionation of A549 and HEK293T cells to determine localization of DUSP11. Cells from one well of 12-well format plate were trypsinized, washed with phosphate-buffered solution (PBS), resuspended in 50-ul of CSKT buffer [10mM PIPES (pH 6.8), 100mM NaCl, 300mM sucrose, 3mM MgCl 2 , 1mM EDTA, 1mM DTT, 0.5% (vol/vol) tritonX-100, protease inhibitors (Roche)], and placed on ice for 10 minutes. Nuclei were then pelleted by centrifugation at 5000xg for 5 minutes. The supernatant [cytosolic fraction (Cyto)] was removed (50-ul) and added to 50-ul of SDS lysis buffer (1% SDS; 2% 2-mercaptoethanol). The nuclei were then washed in 50-ul of CSKT buffer and centrifuged at 5000xg for 5 minutes. The nuclei were then resuspended in 50-ul of water followed by addition of 50-ul of SDS buffer to make the nuclear fraction (Nuc). A whole cell lysate (WCL) was collected in parallel by trypsinization and washing of the cells, which were then resuspended in 50-ul of water, followed by addition of 50-ul of SDS lysis buffer. Samples were boiled for 10 minutes followed by vortexing for 30 seconds. Equal volumes (10-ul) of fractions were then fractionated via 12.5% SDS-PAGE. Protein was then transferred to nitrocellulose membrane (Bio-Rad). Anti-DUSP11 polyclonal rabbit antibody (1:2000 dilution; Proteintech, catalog no. 10204–2-AP), anti-α-Tubulin monoclonal mouse antibody (1:10,000 dilution; Sigma-Aldrich, catalog no. T6199), and anti-Histone H3 (D1H2) rabbit antibody (Cell signaling; mAb #4499) in phosphate-buffered saline (PBS) with 0.1% Tween20 (PBST) and 5% BSA were used to blot for DUSP11, α-Tubulin (cytosolic localized control), and Histone H3 (nuclear localized control). After washing with PBST, membranes were blotted with IRDye 800CW and IRDye <t>680LT</t> secondary antibodies (1:10,000 dilution; LI-COR) in PBST with 5% BSA. Blots were washed 4 times with PBST and then scanned on an Odyssey CLx infrared imaging system (LI-COR). (C) Nuclear/cytosolic fractionation as described in (B) of HEK293T cells (12-well format) transfected with 200-ng/well of the 3xFLAG-tagged DUSP11 constructs from (A). The anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich) was used to stain for the 3xFLAG-tagged DUSP11 proteins. (D) Analysis of the BLV-B2 5p:3p miRNA ratios with co-expression of the DUSP11 constructs from (A). DUSP11-null HEK293T cells (24-well format; 70% confluent) were co-transfected with 5-ng/well of the EBER1 transfection control expression vector (pEBV RIJ), 400-ng/well of pBLV-B2, and 100-ng/well of pDUSP11, pDUSP11-C152, or the DUSP11 construct shown in (A). Northern blot analysis was then performed as described in refs. and . The membrane was probed with the BLV-miR-B2–5p probe, stripped and re-probed with the BLV-miR-B2–3p probe, and stripped and re-probed with the EBER1 probe as a loading/transfection control. Below is immunoblot analysis that was performed on duplicate lysates to confirm and quantitate DUSP11 expression from each construct using the anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich). (E) Bar graph of the relative band density from 4 independent RNA gel blot analyses, as shown in D (except for DUSP11-Δ13–26, in which 3 replicates were performed). Bars represent the average band density ratio (5p/3p) +/− SD of the BLV-B2 miRNAs. The ratio with co-expression of each DUSP11 construct was normalized to the C152S catalytic mutant. Asterisks represent statistical significance (p<0.05) in comparison to wild type DUSP11 (WT). P-values were determined using student's t-test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5229/pmc05785229/pmc05785229__krnb-14-11-1306169-g003.jpg)