Figure 4.

(A) Heat shock treatment of HeLa cells induced an increase in nuclear RNase P-specific activity. (B) Accumulation of Hsp27 in nuclei. (C) tRNA processing after heat shock. HeLa cell culture and heat shock treatment, preparation of nuclear extracts, and assay of RNase P activity are described in Materials and Methods. (A) Assay of RNase P activity in nuclear extracts performed by mixing 1 μg of nuclear extracts with pSupS1 for 5 min at 37°C. (B) Aliquots of 15 μg of nuclear proteins were subjected to 12% SDS/PAGE and transferred to a nitrocellulose filter. The filter was probed with monoclonal antibody against Hsp27 (as shown in B, lane 1, recombinant Hsp27 from E. coli; lanes 2–4: nuclear extracts of non-heat-treated cells or cells heat treated for 1 and 3 h at 43°C, respectively). (C) Total RNA was extracted from heat-treated HeLa cells (heat shock performed at 43°C for 0.5, 1, 2, 4 h) and non-heat-treated cells (harvested after incubation at 37°C for 2 and 4 h as controls). Ten micrograms of total RNA samples was separated on 8% polyacrylamide/7 M urea gel, transferred to nylon membrane, and blotted with a γ-³²P-labeled oligonucleotide complementary to mature tRNA^Leu (Upper), and the same filter was reprobed with a γ-³²P-labeled oligonucleotide that is complementary to human 5S rRNA (Lower). PT, the primary transcript; tRNA, the mature tRNA.