tRNA binds to cytochrome c and inhibits caspase activation - PubMed (original) (raw)

tRNA binds to cytochrome c and inhibits caspase activation

Yide Mei et al. Mol Cell. 2010.

Abstract

The specific molecular events that characterize the intrinsic apoptosis pathway have been the subject of intense research due to the pathway's fundamental role in development, homeostasis, and cancer. This pathway is defined by the release of cytochrome c from mitochondria into the cytosol and subsequent binding of cytochrome c to the caspase activator Apaf-1. Here, we report that both mitochondrial and cytosolic transfer RNA (tRNA) bind to cytochrome c. This binding prevents cytochrome c interaction with Apaf-1, blocking Apaf-1 oligomerization and caspase activation. tRNA hydrolysis in living cells and cell lysates enhances apoptosis and caspase activation, whereas microinjection of tRNA into living cells blocks apoptosis. These findings suggest that tRNA, in addition to its well-established role in gene expression, may determine cellular responsiveness to apoptotic stimuli.

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Figures

Figure 1

Figure 1. RNA hydrolysis enhances cytochrome _c_-induced caspase activation

(A and B) HeLa S100 extracts were incubated with cytochrome c (20 μg/ml) in the presence and absence (−) of increasing amounts of RNase A. (A) Extracts were analyzed by Western blot. Molecular weight (MW) markers (in kilo Daltons) are indicated on the left. C9, procaspase-9; C3, procaspase-3. The levels of actin are shown as a loading control. (B) RNA was separated via PAGE under denaturing conditions and stained with ethidium bromide. (C) Activation of caspase-9 and −3 in HeLa S100 extracts treated with cytochrome c (20 μg/ml), RNase A (30 ng/ml), and RNase inhibitor (In) as indicated. (D) Reticulocyte lysates containing _in vitro_-translated, 35S-labeled procaspase-9 were treated with and without cytochrome c (200 μg/ml) in the presence of the indicated concentration of RNase A. Caspase-9 was detected by autoradiography.

Figure 2

Figure 2. Total cellular RNA inhibits caspase activation in cell extracts and in a reconstituted system

(A) Activation of caspase-9 and −3 in Jurkat S100 extracts treated with cytochrome c (20 μg/ml) alone and in combination with the indicated amounts of RNA. (B) _In vitro_-translated, 35S-labeled procaspase-9 was incubated with purified full-length Apaf-1 (10 nM), dATP (1mM), cytochrome c (20 μg/ml), and increasing amounts of total cellular RNA (0.1, 0.2, and 0.4 μg/μl). Caspase-9 was detected by autoradiography.

Figure 3

Figure 3. RNA interferes with cytochrome c:Apaf-1 interaction and inhibits apoptosome formation

(A) Jurkat S100 extracts were incubated alone (−), with cytochrome c, and with cytochrome c plus RNA at 25 °C for 15 min. Extracts were fractionated on a Superose 6 gel filtration column. The fractions were analyzed by Western blot. The positions of molecular weight standards for the column are marked at the top. (B) His-tagged Apaf-1 bound to Ni-NTA beads was incubated with and without cytochrome c, and with cytochrome c plus increasing amounts of RNA (0.1, 0.2, and 0.4 μg/μl). Bead-bound proteins and one percent of the input were analyzed by Western blot. (C) _In vitro_-translated, 35S-labeled procaspase-9 was incubated with purified Apaf-1Δ, dATP, and increasing amount of RNA as indicated, at 30 °C for 1 h. (D) Jurkat S100 extracts were incubated with cytochrome c for the indicated durations before being analyzed for caspase-9 activation. (E) Jurkat S100 extracts were incubated with and without cytochrome c (lanes 1 and 2), and were pre-incubated with cytochrome c before the addition of tRNA at the indicated time (lanes 3–8). The total reaction time for each sample was 2 h. Activation of caspase-9 was analyzed by Western blot.

Figure 4

Figure 4. Interaction of cytochrome c with tRNA both in vivo and in vitro and the effect of tRNA on caspase-9 activation

(A) Recombinant cytochrome c was incubated with and without total RNA at 25 °C for 15 min. The reaction mixture was loaded on a Superose 6 column and fractions were analyzed by Western blot. (B and C) HeLa cells were treated with formaldehyde, and cell lysates were made in Empigen BB-containing buffer and immunoprecipitated with the indicated antibodies. (B) RNA in the immunoprecipitates was analyzed by Northern blotting using radiolabeled probes for the indicated RNAs. hvg3, human vault RNA; hY1, human Y1 RNA. Input samples contained ~ 1% of the RNA used for IP. (C) Cytochrome c and Smac in immunoprecipitates and ~1.5% of the input were analyzed by Western blot. *, Smac precursor. (D) In vitro synthesized, [32P]UTP-labeled cytosolic (Cy) and mitochondrial (Mt) tRNAs were incubated with increasing amounts (0.5, 2.5, 12.5 μM) of cytochrome c for 45 min at 30 °C. Mixtures were then incubated with 0.5 M Urea (final concentration) for 10 min and analyzed by 6% native gel electrophoresis and autoradiography. Cytochrome c:tRNA complexes are indicated. (E) Radiolabeled total cellular tRNA labeled (12 fmole) was incubated with and without cytochrome c (100 pmol), and with cytochrome c in the presence of non-labeled rRNA, poly(A), total tRNA (50 and 250 ng), and mitochondrial Phe DNA (130 and 520 ng). The mixtures were analyzed by native gel electrophoresis and autoradiography.

Figure 5

Figure 5. tRNA block apoptosome formation and caspase-9 activation

(A) Jurkat S100 extracts were incubated with cytochrome c and increasing amounts of total RNA, rRNA, tRNA (0.1, 0.2, and 0.4 μg/μl), and mRNA (0.02, 0.04, 0.08 μg/μl) at 37 °C for 1 h. Less amounts of mRNA were used because in cells it is expressed at lower levels compared with either tRNA or rRNA. Activation of caspase-9 was analyzed by Western blot. (B) Jurkat S100 extracts were incubated with and without (ctrl) cytochrome c, and with cytochrome c plus tRNA. The extracts were resolved on Superose 6 gel filtration column, and fractions were analyzed by Western blot. (C) Jurkat S100 extracts were incubated with increasing concentrations of cytochrome c in the absence and presence of different concentrations of tRNA for 1 h. Activation of caspase-9 and −3 was analyzed by Western blot.

Figure 6

Figure 6. Microinjection of tRNA blocks cytochrome _c_-induced apoptosis

HEK 293 cells were injected with buffer (ctrl) and the indicated combinations of cytochrome c, zVAD-fmk, tRNA, and rRNA. Dextran-Texas Red (Dex-TR) was included in each injection to label the injected cells. Cells were fixed and stained with DAPI 2 h after injection. (A) Representative images of injected cells. Arrowheads indicate apoptotic cells. Larger images of apoptotic cells are shown in panels d_–_f. (B) Percentage of apoptosis among injected cells. Data presented are the mean ± the standard deviation (SD) of three independent experiments.

Figure 7

Figure 7. Degradation of tRNA enhances apoptosis via the intrinsic pathway

(A) HeLa cells were transfected with 1 μg/ml onconase and cultured for the indicated periods of time. Left: total RNA was separated by 8% urea-containing PAGE and visualized by ethidium bromide staining. Right: activation of caspase-9 and caspase-3, and the cleavage of PARP were analyzed by Western blot. (B) Apaf-1+/+ and _Apaf-1_−/− MEF cells were treated with indicated amounts of onconase for 24 h. Top: percentages of apoptosis are shown as mean ± SD of three independent experiments. Bottom: Apaf-1 expression and PARP cleavage in cell lysates. (C) HeLa cells were transfected with an indicated amount of onconase. 3 h post-transfection, the cells were incubated with and without doxorubicin (Dox, 1μg/ml) for an additional 12 h. Left: percentages of apoptosis. The data represent mean ± SD of three independent experiments. Right: the activation of caspase-9 and −3 and the cleavage of PARP. (D) HeLa cells were transfected with and without onconase (1 μg/ml). 3 h after transfection, cells were treated with Dox (1 μg/ml) for another 12 h. S100 extracts were fractionated on a Superose 6 gel filtration column, and fractions were analyzed by Western blot.

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