Molecular organization of RNP complexes containing P11 antigen in heat-shocked and non-heat-shocked Drosophila cells.
Immunofluorescence analysis of polytene chromosomes of Drosophila melanogaster using the monoclonal antibody P11 has shown that after heat-shock the 38-kDa P11 antigen almost exclusively localizes at heat-shock puff 93D where it is part of giant puff-specific RNP granules. The biochemical experiments reported here show that, independent of growth temperature, the P11 antigen is a component of nuclear 10S RNP particles. The P11-containing 10S snRNPs can be stabilized in CsCl with 20 mM Mg2+ and possess a buoyant density of rho = 1.4 g/cm3. Sucrose gradient analysis of nuclear RNP extracts of heat-shocked Schneider's S-3 tissue culture cells shows that, after a 37 degree C heat-shock, the 10S RNPs associate with large RNP complexes sedimenting at 170-220S. The change in distribution is a temperature-dependent process with intermediate forms at 29 degrees C and 33 degrees C. In thermotolerant cells this observed change in distribution is strongly reduced. DEAE-Sephacel column chromatography and sucrose gradient analysis of nuclear RNP, followed by Northern blot analysis using 93D-specific probes of the TaqI repeat and immunoblotting experiments, show that the P11-containing 10S snRNPs are distinct from the RNP complexes formed by the 93D transcripts, suggesting an indirect association after heat-shock. Our experiments demonstrate that, despite the fact that a 37 degrees C heat-shock does not affect the overall integrity of nuclear RNP, it imposes changes on the general organization and interaction of the nuclear RNP population, resulting in the formation of large nuclear RNP aggregates and complexes. Such changes may be important for the survival strategy of the cell and for hnRNA processing and storage events which are effected by heat-shock.
Schuldt
C
C
Zentrum für Molekulare Biologie und Institut für Molekulare Genetik der Universität Heidelberg, Federal Republic of Germany.
Kloetzel
P M
PM
Bautz
E K
EK
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000302:isolation & purification*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D002875:Chromosomes / Q000032:analysis*; D004331:Drosophila melanogaster; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D008970:Molecular Weight; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D012261:Ribonucleoproteins