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First published online August 26, 2004
doi: 10.1242/10.1242/jcs.01329

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A key role for stress-induced satellite III transcripts in the relocalization of splicing factors into nuclear stress granules

¹ INSERM U309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche CEDEX, France
² Institut de Génétique Moléculaire, CNRS UMR5535, IFR 122, 34293 Montpellier CEDEX 5, France

View larger version (106K): [in a new window]   Fig. 1. HSF1 is the key determinant in recruiting hSF2/ASF and hSRp30c to nuclear stress granules. (A) hSF2/ASF-GFP or hSRp30c-GFP proteins (green) were transiently expressed in HeLa cells and HSF1 (red) was detected by immunofluorescence. Both proteins display a typical speckled pattern at 37°C, and are targeted to HSF1 granules upon stress. (B) hSF2/ASF-GFP or hSRp30c-GFP (green) were co-expressed in HeLa cells with either the HSP70 protein or with a dominant negative mutant of HSF1 (DBD+TRIM-myc) (red). HSP70 and the myc-tagged HSF1 mutant were subsequently detected by immunofluorescence. Overexpression of HSP70 does not alter SR protein distribution at 37°C (left panels). By contrast, at 42°C, HSP70 overexpression prevents the targeting of both hSF2/ASF and hSRp30c to the granules (right panel). Likewise, hSF2/ASF and hSRp30c are not recruited to the granules formed by the DBD+TRIM mutant at 37°C or at 42°C. Bars, 5 µm.

View larger version (73K): [in a new window]   Fig. 2. The second RNA-recognition motif is required for targeting hSF2/AF to the granules. GFP-tagged deletion mutants of hSF2/ASF (green) were transiently expressed in HeLa cells, and their distribution relative to HSF1 (red) was analyzed by immunofluorescence in non heat-shocked and heat-shocked cells. DeletiOn Of tHe RS dOmain (HSF2/ASFRS) or the first RNA-recognition motif (hSF2/ASFRRM1) does not affect the speckled distribution of the protein at 37°C, and does not impede the targeting to nuclear stress granules at 42°C. By contrast, deletion of the second RNA-recognition motif (hSF2/ASFRRM2), which doesnot affect the localization of the protein at 37°C, prevents its stress-induced recruitment to the granules. The RRM2 domain alone is however not sufficient for the targeting to the granules. Bar, 5 µm.

View larger version (36K): [in a new window]   Fig. 3. SR protein targeting to the granules requires the presence of RNA. (A) Transiently expressed hSF2/ASF-GFP protein (green) was detected in heat-shocked HeLa cells together with sat III transcripts (red) and visualized by RNA FISH. hSF2/ASF colocalizes with sat III transcripts within stress granules. Bar: 5 µm. (B) Whether RNA was required in SR protein targeting to the granules was analyzed by treating the cells either with a transcription inhibitor (-amanitin or DRB) added twenty minutes before the end of the 1-hour heat shock, or with RNase A added following heat shock. All three treatments prevent the relocalization of hSF2/ASF and hSRp30c but not of HSF1 and RNA polymerase II to nuclear stress granules. (C) Sat III transcripts co-immunoprecipitate with hSF2/ASF protein. The endogenous hSF2/ASF protein was immunoprecipitated from non heat-shocked or heat-shocked cells with a specific monoclonal antibody (Caceres et al., 1997). Co-immunoprecipitated RNAs were extracted and analyzed by reverse transcription with antisense primers specific for hsp70, hsp90 and sat III transcripts. Sense primers to hsp90 transcripts were used as a negative control. The y-axis corresponds to the intensity ratios between signal and input. In non heat-shocked cells kept at 37°C, a strong signal for the constitutively expressed hsp90 transcripts and faint signals for hsp70 and sat III transcripts are observed. In cells that were heat-shocked for one hour at 42°C or that were allowed to recover for 3 hours at 37°C following heat shock (rec), a strong signal is obtained for all three transcripts, thus showing that they were all present in vivo in a complex with hSF2/ASF. As expected, no significant signal was obtained with the sense primer to hsp90 transcripts.

View larger version (46K): [in a new window]   Fig. 4. Sat III transcripts are associated with Sm proteins. (A) SnRNPs (green) were detected in non heat-schocked and heat-shocked HeLa cells together with HSF1 (red). SnRNPs were not recruited to the stress granules although they were not excluded from these regions. Bar: 5 µm. (B) The endogenous snRNPs proteins were immunoprecipitated from non heat-shocked or heat-shocked cells with a specific monoclonal antibody (clone Y12). Co-immunoprecipitated RNAs were extracted and analyzed by reverse transcription as previously described. In non heat-shocked cells (37°C), a strong signal for the constitutively expressed hsp90 transcripts and faint signals for hsp70 and sat III transcripts were observed. In cells that were heat-shocked for 1 hour at 42°C or that were allowed to recover for 3 hours at 37°C following heat shock (rec), a strong signal is obtained for hsp90 and sat III transcripts but not for the intronless hsp70 transcripts, thus showing that hsp90 and sat III transcripts are present in vivo in a complex with snRNPs. As expected, no significant signal was obtained with the sense primer to hsp90 transcripts.

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