Effect of heat shock on RNA metabolism in HeLa cells

Sadis, Seth; Hickey, Eileen; Weber, Lee A.

doi:10.1002/jcp.1041350304

Cited by 71 publications

(39 citation statements)

References 55 publications

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“…This finding is consistent with results of the recent study by Sadis et al (42), who also examined heat shock in HeLa cells; they propose that the decreased accumulation is due to instability of the newly synthesized rRNAs. One possible explanation for the apparent discrepancy in the amounts of newly synthesized rRNA after heat shock in human cells may be related to the fact that both our study and that of Sadis et al (42) analyzed rRNAs shortly (1 to 4 h) after induction of the heat shock, whereas in the earlier study (47) the stress was inflicted upon the cells for at least 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 It has recently become clear that different stresses that induce HSPs can vary in their effects. For example, Bond found that heat shock and sodium arsenite treatment had differing effects on pre-mRNA splicing (8).…”

Section: Discussionsupporting

confidence: 93%

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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Human rRNA precursors from normal or stressed HeLa cells were studied by Si nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with[32p]PO4. Heating cells to 43°C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42°C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43°C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

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Section: Discussionsupporting

confidence: 93%

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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“…Release of nucleolin from the nucleolus can be mediated by a loss of its pre-rRNA binding substrate, which occurs in heated cells as heat exposure decreases rRNA synthesis (68,69). However, because nucleolin is a phosphoprotein that can be modified by several kinases including Cdc2, protein kinase C , and casein kinase II (56,57), and can also be methylated and ADP-ribosylated, it is possible that its translocation into the nucleoplasm is the result of post-translational modifications.…”

Section: Effects Of Heat On Nucleolin Localization In Vivo-mentioning

confidence: 99%

Regulation of DNA Replication after Heat Shock by Replication Protein A-Nucleolin Interactions

Wang

Guan

Wang

et al. 2001

Journal of Biological Chemistry

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Heat shock inhibits replicative DNA synthesis, but the underlying mechanism remains unknown. We investigated mechanistic aspects of this regulation in melanoma cells using a simian virus 40 (SV40)-based in vitro DNA replication assay. Heat shock (44°C) caused a monotonic inhibition of cellular DNA replication following exposures for 5-90 min. SV40 DNA replication activity in extracts of similarly heated cells also decreased after 5-30 min of exposure, but returned to near control levels after 60 -90 min of exposure. This transient inhibition of SV40 DNA replication was eliminated by recombinant replication protein A (rRPA), suggesting a regulatory process targeting this key DNA replication factor. SV40 DNA replication inhibition was associated with a transient increase in the interaction between nucleolin and RPA that peaked at 20 -30 min. Because binding to nucleolin compromises the ability of RPA to support SV40 DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock. The relevance of this interaction to the regulation of cellular DNA replication is indicated by the transient translocation in heated cells of nucleolin from the nucleolus into the nucleoplasm with kinetics very similar to those of SV40 DNA replication inhibition and of RPA-nucleolin interaction. Because the targeting of RPA by nucleolin in heated cells occurs in an environment that preserves the activity of several essential DNA replication factors, active processes may contribute to DNA replication inhibition to a larger degree than presently thought. RPAnucleolin interactions may reflect an early step in the regulation of DNA replication, as nucleolin relocalized into the nucleolus 1-2 h after heat exposure but cellular DNA replication remained inhibited for up to 8 h. We propose that the nucleolus functions as a heat sensor that uses nucleolin as a signaling molecule to initiate inhibitory responses equivalent to a checkpoint.DNA replication is at the center of the eukaryotic cell cycle, and its regulation under conditions of stress, or after exposure to DNA-damaging agents, is under the control of intricate pathways that are only now beginning to be characterized. Although the mechanisms regulating DNA replication after DNA damage are studied intensively and a considerable amount of information is now available (reviewed in Ref.

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“…Heat shock is known to inhibit RNA transcription and mRNA processing (26,37), while low doses of ionizing radiation have relatively little affect. We investigated whether the slower rate of topoisomerase II␣ mRNA degradation in cells following irradiation could be accounted for by ongoing transcription.…”

Section: Vol 16 1996 Cell Cycle-regulated Expression Of Topoisomeramentioning

confidence: 99%

The Cell Cycle-Coupled Expression of Topoisomerase IIα during S Phase Is Regulated by mRNA Stability and Is Disrupted by Heat Shock or Ionizing Radiation

Goswami

Roti

Hunt

1996

Molecular and Cellular Biology

111

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Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase II␣ and II␤ genes. In HeLa cells synchronized by mitotic shake-off, topoisomerase II␣ mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G 1 phase. Also detected was a corresponding increase in topoisomerase II␣ protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G 2 phases. Topoisomerase II␣ expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase II␣ expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, a half-life of greater than 4 h was observed. However, during G 1 phase, when cellular levels are lowest, the half-life of topoisomerase II␣ mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase II␣ mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45؇C for 15 min) caused the accumulated topoisomerase II␣ mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase II␣ expression and cell cycle progression delays in late S/G 2 .The double-stranded nature of the DNA molecule presents special problems during chromosomal replication and segregation. Movement of the replication fork along the DNA molecule generates positive supercoiling in advance while the replicated parental strands in its wake are negatively supercoiled. Topological problems also arise near the end of replication when adjacent replication forks converge. Depending on the relative unwinding rate of the parental or progeny strands, either an intertwined or a gapped structure is formed (47). Unless untangled, the former structure leads to chromosome breakage, nondisjunction, and cell death (20). DNA topology in the cell is controlled through the action of two types of topoisomerase enzymes (see references 20 and 46 for reviews). Type I topoisomerases introduce into the duplex DNA molecule transient single-strand nicks which act as swivels during DNA replication or relieve the torsional stress associated with RNA transcription. Topoisomerase II enzymes relax, catenate, or decatenate DNA by introducing transient double-strand breaks which allow another DNA segment to pass through. While topoisomerase II is also capable, in theory, of relaxing transcription and replication induced supercoiling, it appears that topoisomerase I is mainly responsible for ...

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Effect of heat shock on RNA metabolism in HeLa cells

Cited by 71 publications

References 55 publications

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Regulation of DNA Replication after Heat Shock by Replication Protein A-Nucleolin Interactions

The Cell Cycle-Coupled Expression of Topoisomerase IIα during S Phase Is Regulated by mRNA Stability and Is Disrupted by Heat Shock or Ionizing Radiation

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