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Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3' Uridylated Intermediates Degraded by DIS3L2.

Thomas MP, Liu X, Whangbo J, McCrossan G, Sanborn KB, Basar E, Walch M, Lieberman J - Cell Rep (2015)

Bottom Line: Apoptosis is a tightly coordinated cell death program that damages mitochondria, DNA, proteins, and membrane lipids.Little is known about the fate of RNA as cells die.Our results suggest that global mRNA decay is an overlooked hallmark of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.

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mRNA Decay Begins Early in Cell Death and Requires MOMP(A–E) Jurkat cells were treated with ActD ± αFas or arsenite for 4 hr and harvested hourly for qRT-PCR (A), immunoblot (B), caspase activity by luminescent assay (AU, arbitrary units) (C), annexin V staining by flow cytometry (D), and DNA fragmentation by electrophoresis (E). Asterisks in (B) indicate caspase cleavage products. mRNA decay was concurrent with caspase activation, but preceded DNA cleavage and phosphatidylserine externalization. Error bars represent the SEM of at least three independent experiments. Results in (B) and (E) are representative of multiple experiments, and the experiment shown in (C) was performed once.(F) Jurkat cells were treated for 1 hr with ActD ± αFas and stained with DiIC1(5) to measure mitochondrial depolarization. The transmembrane potential began dissipating within 1 hr after αFas treatment. Results are representative of at least three independent experiments.(G and H) HeLa-BCL2 and HeLa-puro were treated for 6 hr with STS ± zVAD.(G) Fractionated cells were analyzed by immunoblot (top) for cytochrome c release. Effector caspase activity was measured by luminescent reporter assay (bottom). Error bars represent the SEM of separate wells from one experiment.(H) RNAs were assayed by qRT-PCR. Error bars represent the SEM of at least three independent experiments (*p < 0.05). Results were normalized to untreated cells. BCL2 overexpression inhibited mRNA decay to a greater extent than did zVAD. Survival (assayed by CytoTox-Glo assay performed after 6 hr of STS treatment followed by a 24 hr recovery period) was normalized to untreated cells. See also Figure S3.
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Figure 3: mRNA Decay Begins Early in Cell Death and Requires MOMP(A–E) Jurkat cells were treated with ActD ± αFas or arsenite for 4 hr and harvested hourly for qRT-PCR (A), immunoblot (B), caspase activity by luminescent assay (AU, arbitrary units) (C), annexin V staining by flow cytometry (D), and DNA fragmentation by electrophoresis (E). Asterisks in (B) indicate caspase cleavage products. mRNA decay was concurrent with caspase activation, but preceded DNA cleavage and phosphatidylserine externalization. Error bars represent the SEM of at least three independent experiments. Results in (B) and (E) are representative of multiple experiments, and the experiment shown in (C) was performed once.(F) Jurkat cells were treated for 1 hr with ActD ± αFas and stained with DiIC1(5) to measure mitochondrial depolarization. The transmembrane potential began dissipating within 1 hr after αFas treatment. Results are representative of at least three independent experiments.(G and H) HeLa-BCL2 and HeLa-puro were treated for 6 hr with STS ± zVAD.(G) Fractionated cells were analyzed by immunoblot (top) for cytochrome c release. Effector caspase activity was measured by luminescent reporter assay (bottom). Error bars represent the SEM of separate wells from one experiment.(H) RNAs were assayed by qRT-PCR. Error bars represent the SEM of at least three independent experiments (*p < 0.05). Results were normalized to untreated cells. BCL2 overexpression inhibited mRNA decay to a greater extent than did zVAD. Survival (assayed by CytoTox-Glo assay performed after 6 hr of STS treatment followed by a 24 hr recovery period) was normalized to untreated cells. See also Figure S3.

Mentions: We next determined the kinetics of the mRNA decline relative to hallmarks of apoptosis. Jurkat cells treated with ActD ± αFas were harvested hourly over 4 hr and analyzed for housekeeping gene mRNA stability by qRT-PCR, cleavage of PARP-1 and eiF4G by immunoblot, phosphorylation of eiF2α by immunoblot, caspase activation by a luminescent assay, annexin V binding, and DNA fragmentation (Figures 3A–3E). ACTB and GAPDH mRNAs began to decline within 1 hr after addition of αFas and leveled off by 3 hr, whereas 7SL ncRNA was stable (Figure 3A). Nonapoptotic oxidative stress caused by arsenite (+ActD) and caspase-independent programmed cell death induced by the killer protease granzyme A did not affect mRNA levels (Figure 3A and data not shown). Caspase activation and PARP-1 cleavage began at 1 hr and were complete by 3 hr (Figures 3B and 3C). eIF2α phosphorylation and eIF4G cleavage were first detected after 2 hr (Figure 3B). Similarly, phosphatidylserine externalization and DNA fragmentation were not detected until 2 hr after αFas was added (Figures 3D and 3E). Mitochondrial depolarization began within 1 hr after αFas treatment (Figure 3F). Thus, mRNA decay occurred early in cell death coincidently with caspase activation and mitochondrial disruption.


Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3' Uridylated Intermediates Degraded by DIS3L2.

Thomas MP, Liu X, Whangbo J, McCrossan G, Sanborn KB, Basar E, Walch M, Lieberman J - Cell Rep (2015)

mRNA Decay Begins Early in Cell Death and Requires MOMP(A–E) Jurkat cells were treated with ActD ± αFas or arsenite for 4 hr and harvested hourly for qRT-PCR (A), immunoblot (B), caspase activity by luminescent assay (AU, arbitrary units) (C), annexin V staining by flow cytometry (D), and DNA fragmentation by electrophoresis (E). Asterisks in (B) indicate caspase cleavage products. mRNA decay was concurrent with caspase activation, but preceded DNA cleavage and phosphatidylserine externalization. Error bars represent the SEM of at least three independent experiments. Results in (B) and (E) are representative of multiple experiments, and the experiment shown in (C) was performed once.(F) Jurkat cells were treated for 1 hr with ActD ± αFas and stained with DiIC1(5) to measure mitochondrial depolarization. The transmembrane potential began dissipating within 1 hr after αFas treatment. Results are representative of at least three independent experiments.(G and H) HeLa-BCL2 and HeLa-puro were treated for 6 hr with STS ± zVAD.(G) Fractionated cells were analyzed by immunoblot (top) for cytochrome c release. Effector caspase activity was measured by luminescent reporter assay (bottom). Error bars represent the SEM of separate wells from one experiment.(H) RNAs were assayed by qRT-PCR. Error bars represent the SEM of at least three independent experiments (*p < 0.05). Results were normalized to untreated cells. BCL2 overexpression inhibited mRNA decay to a greater extent than did zVAD. Survival (assayed by CytoTox-Glo assay performed after 6 hr of STS treatment followed by a 24 hr recovery period) was normalized to untreated cells. See also Figure S3.
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Figure 3: mRNA Decay Begins Early in Cell Death and Requires MOMP(A–E) Jurkat cells were treated with ActD ± αFas or arsenite for 4 hr and harvested hourly for qRT-PCR (A), immunoblot (B), caspase activity by luminescent assay (AU, arbitrary units) (C), annexin V staining by flow cytometry (D), and DNA fragmentation by electrophoresis (E). Asterisks in (B) indicate caspase cleavage products. mRNA decay was concurrent with caspase activation, but preceded DNA cleavage and phosphatidylserine externalization. Error bars represent the SEM of at least three independent experiments. Results in (B) and (E) are representative of multiple experiments, and the experiment shown in (C) was performed once.(F) Jurkat cells were treated for 1 hr with ActD ± αFas and stained with DiIC1(5) to measure mitochondrial depolarization. The transmembrane potential began dissipating within 1 hr after αFas treatment. Results are representative of at least three independent experiments.(G and H) HeLa-BCL2 and HeLa-puro were treated for 6 hr with STS ± zVAD.(G) Fractionated cells were analyzed by immunoblot (top) for cytochrome c release. Effector caspase activity was measured by luminescent reporter assay (bottom). Error bars represent the SEM of separate wells from one experiment.(H) RNAs were assayed by qRT-PCR. Error bars represent the SEM of at least three independent experiments (*p < 0.05). Results were normalized to untreated cells. BCL2 overexpression inhibited mRNA decay to a greater extent than did zVAD. Survival (assayed by CytoTox-Glo assay performed after 6 hr of STS treatment followed by a 24 hr recovery period) was normalized to untreated cells. See also Figure S3.
Mentions: We next determined the kinetics of the mRNA decline relative to hallmarks of apoptosis. Jurkat cells treated with ActD ± αFas were harvested hourly over 4 hr and analyzed for housekeeping gene mRNA stability by qRT-PCR, cleavage of PARP-1 and eiF4G by immunoblot, phosphorylation of eiF2α by immunoblot, caspase activation by a luminescent assay, annexin V binding, and DNA fragmentation (Figures 3A–3E). ACTB and GAPDH mRNAs began to decline within 1 hr after addition of αFas and leveled off by 3 hr, whereas 7SL ncRNA was stable (Figure 3A). Nonapoptotic oxidative stress caused by arsenite (+ActD) and caspase-independent programmed cell death induced by the killer protease granzyme A did not affect mRNA levels (Figure 3A and data not shown). Caspase activation and PARP-1 cleavage began at 1 hr and were complete by 3 hr (Figures 3B and 3C). eIF2α phosphorylation and eIF4G cleavage were first detected after 2 hr (Figure 3B). Similarly, phosphatidylserine externalization and DNA fragmentation were not detected until 2 hr after αFas was added (Figures 3D and 3E). Mitochondrial depolarization began within 1 hr after αFas treatment (Figure 3F). Thus, mRNA decay occurred early in cell death coincidently with caspase activation and mitochondrial disruption.

Bottom Line: Apoptosis is a tightly coordinated cell death program that damages mitochondria, DNA, proteins, and membrane lipids.Little is known about the fate of RNA as cells die.Our results suggest that global mRNA decay is an overlooked hallmark of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.

Show MeSH
Related in: MedlinePlus