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First published online 22 February 2005
doi: 10.1242/jcs.01720

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Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing

Noam Shomron^*, Moti Alberstein^*, Mika Reznik and Gil Ast

Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel

View larger version (38K): [in a new window]   Fig. 1. Stress induces cytoplasmic hSlu7 accumulation. (A) HeLa cells were mock treated (i), incubated at 42°C for 1 hour (ii) or irradiated with UV-C (0.3 J cm–2 or 0.9 J cm–2 as indicated on the left; iii-v). Following a 3 hour recovery period under optimal conditions, cells were fixed and hSlu7 cellular localization was observed by indirect immunofluorescence using anti-hSlu7 antibody. (left) Nuclear DNA DAPI staining; (center) anti-hSlu7 staining; (right) merged image of the two. (B) As described in A except that the cells were fixed and snRNP cellular localization was observed by indirect immunofluorescence using anti-Sm antibody. Treatments are indicated on the left. (C) As described in A except that the cells were incubated in the presence of actinomycin-D (ActD; 5 µg ml–1) or puromycin (Puro; 300 ng ml–1) for 3 hours before UV-C irradiation (UV-C). (D, left) Time-dependent export of hSlu7 out of the nucleus following irradiation at 0.3 J cm–2. The y-axis indicates the proportion of hSlu7 exported out of the nucleus. (D, right) The effects of various stress conditions on the export of hSlu7 out of the nucleus. HeLa cells were subjected to various stress conditions [UV-C (0.3 J cm–2), heat (42°C for 1 hour), H2O2, sorbitol (OSM), cisplatin (cisP) and Neocarzinostatin (NCS)]. 3 hours later, the cellular localization of hSlu7 was observed by indirect immunofluorescence using anti-hSlu7. The error bars represent s.d.

View larger version (38K): [in a new window]   Fig. 2. Alternative splicing and 3'ss selection are affected by hSlu7 nuclear concentration. (A) The ADAR2 minigenes and the sequence of the Alu exon 3'ss. The selected 3'ss is underlined. Proximal (left) and distal (right) AGs are indicated in bold. The 3'ss of the intron-2/exon-3 junction of the gene encoding D-aspartate oxidase (DDO) is shown at the bottom. Lower- and upper-case letters represent intron and exon sequences, respectively. (B) Cells were transfected with either of the ADAR2 mini-genes (ADAR2 and ADAR2+10) (Lev-Maor et al., 2003). Forty-eight hours after transfection, cells were either mock treated or UV-C irradiated and then incubated for 3 hours at optimal conditions. Then, cytoplasmic RNA was collected followed by RT-PCR using specific primers and separation on 2% agarose gel (lanes 2-9). Some of the cells were co-transfected with a plasmid for RNAi directed against hSlu7 (RNAi) or a control plasmid (Plasmid; lanes 6-9). A schematic representation of PCR products is indicated to the right of the gel. The alternative Alu exon is colored black. GAPDH, a house-keeping gene, was amplified from each sample to confirm that approximately the same amount of cDNA was used for each reaction. (C) A graph quantifying the spliced products, including exon 8 from B, expressed out of the total amount of spliced products in the same lane and experiment. The error bars represent s.d. (D) Cells were transfected with either an RNAi plasmid directed against hSlu7 or a control plasmid. After 1 day or 2 days, total proteins were extracted, separated by 12% SDS-PAGE, transferred to a membrane and probed for hSlu7, hPrp18 and GAPDH. Lane 1: `No trans.' indicates cells that were not transfected. Lane 2: `Plasmid' indicates cells transfected with a control plasmid. Lanes 3 and 4, `RNAi' indicates cells transfected with an RNAi plasmid directed against hSlu7 for 1 day (1d) or 2 days (2d), respectively. The 55 kDa protein is a non-specific interaction of anti-hPrp18 antibody. (E) Cells were either mock treated (lane 2) or UV-C irradiated (lane 3), then incubated for 3 hours at optimal conditions followed by RNA isolation and RT-PCR. Some of the cells were transfected with an RNAi plasmid directed against hSlu7 (RNAi; lane 5) or a control plasmid (Plasmid; lane 4). A schematic representation of PCR products is indicated to the right of the gel. (F) A graph quantifying the spliced products including/excluding the alternative DDO exon from E expressed out of the total amount of spliced products in the same lane and experiment.

View larger version (20K): [in a new window]   Fig. 3. hSlu7 effect is specific to 3'ss selection. (A) The E1A pre-mRNA containing three alternative 5'ss (9S, 12S and 13S, from distal to proximal). The 10S product is produced by aberrant splicing that does not involve simple competition between alternative 5'ss (Stephens and Harlow, 1987). (B) Spliced products were analysed following RT-PCR using primers on either side of the alternative exon (A, arrows). (bottom) RT-PCR carried out on a control GAPDH gene. (C) The relative amounts of 9S, 12S and 13S used in B expressed out of the total amount of spliced products in the same lane and experiment.

View larger version (46K): [in a new window]   Fig. 4. The JNK signaling pathway might be responsible for the subcellular distribution of hSlu7. (A) HeLa cells were incubated with either SB205823 (an inhibitor of the p38 pathway; p38-I) or SP600125 (an inhibitor of the JNK pathway; JNK-I) for 1 hour at the indicated concentrations (bottom right corner of each image; units are in µM). Cells were then UV-C irradiated (0.9 J cm–2) and allowed to recover for 3 hours, followed by fixation and staining with anti-hSlu7. (B) Proportion of hSlu7 exported out of the nucleus. Inhibitor concentration (low, high and medium) refers to 5 µM, 10 µM and 40 µM for SB205823 (p38 inhibitor; shaded bars) and to 0.1 µM, 1 µM and 10 µM for SP600125 (JNK inhibitor; white bars). (C) Inhibition of p38 and JNK phosphorylation by SB205823 and SP600125, respectively, is effective. HeLa cells were UV-C irradiated (0.9 J cm–2) and then allowed to recover for 1 hour or 3 hours, as indicated (lanes 1,2 and 3,4, respectively). To some cells, either SB205823 (40 µM; p38-I; top) or SP600125 (10 µM; JNK-I; bottom) was added. Total proteins were extracted, separated by SDS-PAGE and probed with a monoclonal anti-phospho-p38-specific antibody (p38-P) or a polyclonal anti-phospho-Jun-specific antibody (JNK-P). (D) Inhibition of JNK phosphorylation reverses the UV-C irradiation effect on DDO alternative splicing. Cells were mock treated (lane 2) or UV-C irradiated (lanes 3,4) and then incubated for 24 hours at optimal conditions followed by RNA isolation and RT-PCR. One sample was pretreated with 1 µM SP600125 (JNK inhibitor; JNK-I) for 1 hour before irradiation (lane 4). (E) Active JNK1 affects DDO alternative splicing. Cells were mock treated (lane 2) or UV-C irradiated at a low dose (0.01 J cm–2; lanes 3,4) in the absence or presence of a JNK1-cDNA-containing plasmid (lanes 3,4, respectively). Following 24 hours at optimal conditions, RNA was isolation and RT-PCR performed. (F) Active JNK1 increases the hSlu7 nuclear to cytoplasmic shift. 293T cells were either UV-C irradiated (i) or transfected with a JNK1-cDNA-containing plasmid (ii; detected using an HA tag) and then UV-C irradiated at a low dose (0.01 J Cm–2; iii). hSlu7 and JNK1 cellular localization was observed by indirect immunofluorescence by fixing the cells and using anti-hSlu7 and anti-HA antibodies. Superimposed images are presented where nuclear DNA DAPI staining is colored blue (i-iii), anti-hSlu7 staining is colored red (i and iii) and anti-HA-tag (fused to the JNK1 cDNA; ii) is colored green.

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