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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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ZCCHC6/ZCCHC11 Knockdown Inhibits Cell Death and mRNA Decay(A) HeLa cells were transfected with control (CTL), ZCCHC6, and/or ZCCHC11 or PAPD7 siRNAs and harvested 72 hr later. RNA was then analyzed by qRT-PCR to assess knockdown.(B) Accumulation of uridylated decay intermediates was assessed by RT-PCR with an A12-adaptor primer (as in Figure 5F). Uridylated intermediates that arose after STS treatment were reduced after knockdown of ZCCHC6 and/or ZCCHC11. The arrowhead denotes new products and the asterisk denotes products primed from oligouridylate tracts in the ACTB 3′ UTR. Results are representative of three independent experiments.(C–F) After knockdown and STS treatment, mRNA levels were assayed by qRT-PCR (C). Cells were analyzed for cell death by annexin V staining and flow cytometry (D). Caspase activation was assessed by immunoblot for caspase 3 (E) and a luminescent caspase activity assay (F). ZCCHC6 and/or ZCCHC11 knockdown partially restored mRNA levels, rescued cell death, and reduced caspase 3 cleavage and activation. Error bars represent the SEM of at least three independent experiments.**p < 0.01; ***p < 0.001, relative to CTL knockdown.
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Figure 6: ZCCHC6/ZCCHC11 Knockdown Inhibits Cell Death and mRNA Decay(A) HeLa cells were transfected with control (CTL), ZCCHC6, and/or ZCCHC11 or PAPD7 siRNAs and harvested 72 hr later. RNA was then analyzed by qRT-PCR to assess knockdown.(B) Accumulation of uridylated decay intermediates was assessed by RT-PCR with an A12-adaptor primer (as in Figure 5F). Uridylated intermediates that arose after STS treatment were reduced after knockdown of ZCCHC6 and/or ZCCHC11. The arrowhead denotes new products and the asterisk denotes products primed from oligouridylate tracts in the ACTB 3′ UTR. Results are representative of three independent experiments.(C–F) After knockdown and STS treatment, mRNA levels were assayed by qRT-PCR (C). Cells were analyzed for cell death by annexin V staining and flow cytometry (D). Caspase activation was assessed by immunoblot for caspase 3 (E) and a luminescent caspase activity assay (F). ZCCHC6 and/or ZCCHC11 knockdown partially restored mRNA levels, rescued cell death, and reduced caspase 3 cleavage and activation. Error bars represent the SEM of at least three independent experiments.**p < 0.01; ***p < 0.001, relative to CTL knockdown.

Mentions: We hypothesized that one or more TUTases add these non-templated uridylate-rich tails. We focused on ZCCHC6 and ZCCHC11 because they uridylate other cytosolic RNAs (Schmidt et al., 2011; Thornton et al., 2012, 2014). We transfected HeLa cells with a control siRNA or siRNAs targeting ZCCHC6 and/or ZCCHC11, or the TUTase PAPD7 (Figure 6A). Knockdown of ZCCHC6, ZCCHC11, or both reduced apoptotic 3′ uridylation of the ACTB mRNA (Figure 6B) and partially rescued mRNA levels after STS treatment (Figure 6C), whereas PAPD7 knock-down had no effect. ZCCHC6 and ZCCHC11 siRNAs, alone and in combination, reduced annexin V staining (Figure 6D) and caspase 3 cleavage and activation (Figures 6E and 6F) in response to STS. Again, PAPD7 siRNAs had no effect. Collectively, these results suggest that ZCCHC6 and ZCCHC11 collaborate to uridylate mRNAs during cell death, promoting mRNA decay and apoptosis.


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)

ZCCHC6/ZCCHC11 Knockdown Inhibits Cell Death and mRNA Decay(A) HeLa cells were transfected with control (CTL), ZCCHC6, and/or ZCCHC11 or PAPD7 siRNAs and harvested 72 hr later. RNA was then analyzed by qRT-PCR to assess knockdown.(B) Accumulation of uridylated decay intermediates was assessed by RT-PCR with an A12-adaptor primer (as in Figure 5F). Uridylated intermediates that arose after STS treatment were reduced after knockdown of ZCCHC6 and/or ZCCHC11. The arrowhead denotes new products and the asterisk denotes products primed from oligouridylate tracts in the ACTB 3′ UTR. Results are representative of three independent experiments.(C–F) After knockdown and STS treatment, mRNA levels were assayed by qRT-PCR (C). Cells were analyzed for cell death by annexin V staining and flow cytometry (D). Caspase activation was assessed by immunoblot for caspase 3 (E) and a luminescent caspase activity assay (F). ZCCHC6 and/or ZCCHC11 knockdown partially restored mRNA levels, rescued cell death, and reduced caspase 3 cleavage and activation. Error bars represent the SEM of at least three independent experiments.**p < 0.01; ***p < 0.001, relative to CTL knockdown.
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Figure 6: ZCCHC6/ZCCHC11 Knockdown Inhibits Cell Death and mRNA Decay(A) HeLa cells were transfected with control (CTL), ZCCHC6, and/or ZCCHC11 or PAPD7 siRNAs and harvested 72 hr later. RNA was then analyzed by qRT-PCR to assess knockdown.(B) Accumulation of uridylated decay intermediates was assessed by RT-PCR with an A12-adaptor primer (as in Figure 5F). Uridylated intermediates that arose after STS treatment were reduced after knockdown of ZCCHC6 and/or ZCCHC11. The arrowhead denotes new products and the asterisk denotes products primed from oligouridylate tracts in the ACTB 3′ UTR. Results are representative of three independent experiments.(C–F) After knockdown and STS treatment, mRNA levels were assayed by qRT-PCR (C). Cells were analyzed for cell death by annexin V staining and flow cytometry (D). Caspase activation was assessed by immunoblot for caspase 3 (E) and a luminescent caspase activity assay (F). ZCCHC6 and/or ZCCHC11 knockdown partially restored mRNA levels, rescued cell death, and reduced caspase 3 cleavage and activation. Error bars represent the SEM of at least three independent experiments.**p < 0.01; ***p < 0.001, relative to CTL knockdown.
Mentions: We hypothesized that one or more TUTases add these non-templated uridylate-rich tails. We focused on ZCCHC6 and ZCCHC11 because they uridylate other cytosolic RNAs (Schmidt et al., 2011; Thornton et al., 2012, 2014). We transfected HeLa cells with a control siRNA or siRNAs targeting ZCCHC6 and/or ZCCHC11, or the TUTase PAPD7 (Figure 6A). Knockdown of ZCCHC6, ZCCHC11, or both reduced apoptotic 3′ uridylation of the ACTB mRNA (Figure 6B) and partially rescued mRNA levels after STS treatment (Figure 6C), whereas PAPD7 knock-down had no effect. ZCCHC6 and ZCCHC11 siRNAs, alone and in combination, reduced annexin V staining (Figure 6D) and caspase 3 cleavage and activation (Figures 6E and 6F) in response to STS. Again, PAPD7 siRNAs had no effect. Collectively, these results suggest that ZCCHC6 and ZCCHC11 collaborate to uridylate mRNAs during cell death, promoting mRNA decay and apoptosis.

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