Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3' Uridylated Intermediates Degraded by DIS3L2.
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.
Affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.Show MeSH
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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.
Affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.