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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.
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