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Degradation of cellular mRNA is a general early apoptosis-induced event¹

M. JULIETA DEL PRETE^*, MARIA S. ROBLES^*, ANA GUÍO^*,2, CARLOS MARTÍNEZ-A^*, MANUEL IZQUIERDO^*, and JOSE A. GARCIA-SANZ^*3

^* Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco de la UAM, E-28049, Madrid, Spain; and
Instituto de Biología y Genética Molecular (IBGM-CSIC), Facultad de Medicina Universidad de Valladolid, Ramón y Cajal 7, E-47005 Valladolid, Spain

³Correspondence: Department of Immunology and Oncology Centro Nacional de Biotecnología (CNB-CSIC) Campus de Cantoblanco UAM, E-28049 Madrid, Spain. E-mail: jasanz{at}cnb.uam.es

SPECIFIC AIMS

Apoptosis plays a key role during development and homeostasis in multicellular organisms, since aberrant apoptosis leads to disease. Caspase activation, one of the common features of this process, proteolysis of cellular substrates, chromatin condensation, and DNA cleavage by the caspase-activated DNase (CAD) are some of the apoptotic features conserved during evolution. The aim of the present study was to analyze the fate of cytoplasmic mRNAs during the onset of apoptosis.

PRINCIPAL FINDINGS

1. Cytoplasmic mRNAs are actively degraded during apoptosis, independent of the cell-type and the apoptosis-triggering pathway
The fate of cytoplasmic mRNA during apoptosis was analyzed using several cell lines in which apoptosis was induced by a variety of signals (anti-FAS mAb, activation induced cell death [AICD], growth factor deprivation, or okadaic acid). The onset of apoptosis was monitored by exposure of phosphatidyl-serine to the outer surface of the cell membrane (annexin V binding), loss of genomic DNA (appearance of cells in subG₀/G₁), or poly-ADP-ribose polymerase (PARP) processing. Analysis of cells during apoptosis demonstrates that cytoplasmic mRNA degradation occurs as an early apoptotic event, independent of the apoptotic signal and the cell line used.

AICD allows us to analyze the fate of RNAs expressed in a constitutive (TCRß, HLA-I, ß-actin, and 28S rRNA) or inducible fashion (early activation marker CD69). Concurring with the results of experiments in which apoptosis was induced with either anti-CD95 mAb, growth factor deprivation, or okadaic acid treatment, all analyzed RNAs were rapidly degraded on AICD (Fig. 1 A). Specificity was demonstrated by mixing experiments in which an aliquot of apoptotic cells was split into two halves: cytoplasmic RNA was prepared directly from one half (-A20), the other was mixed with mouse control cells (+A20) before lysis and cytoplasmic RNA purification. mRNAs from the apoptotic cells were degraded in both samples (-A20,+A20) whereas the A20-specific mRNA (IA) remained intact (Fig. 1) . The kinetics of apoptosis-induced mRNA degradation correlated with the appearance of the early apoptotic marker annexin V (Fig. 1B ). The apparent difference in ß-actin degradation kinetics between samples containing Jurkat mRNAs alone or mixed with A20 can be explained by the cross-reactivity of this probe between mouse and human samples (Fig. 1) . The 28S rRNA probe cross-reacts with mouse and human samples, but degradation of this rRNA could be quantified by appearance of specific degradation products (Fig. 1A ), which show a similar kinetics in samples containing or not control A20 cells (Fig. 1B , inset).

View larger version (75K): [in this window] [in a new window]   Figure 1. Apoptosis-induced cell death triggers mRNA degradation. After induction of AICD by carbachol in Jurkat JHM12 cells, the kinetics of cytoplasmic mRNA degradation was analyzed. A) Methylene blue staining of cytoplasmic RNA from Jurkat cells alone (-A20) or in combination with control cells (+A20) (staining, top panel). Relative expression levels are shown for 28S rRNA, TCRß, HLA-I, ß-actin, and CD69 in Jurkat cells, as well as for the A20 cell-specific IA mRNA. Open arrowheads indicate 28S rRNA degradation products. B) Quantification of data in panel A, including the control IA mRNA, as percentage of the remaining mRNA and fraction of cells positive for annexin V, which for comparative purposes is represented as (100–% annexin V). Inset: appearance kinetics of 28S rRNA degradation product (open arrowhead in 28S rRNA panel of panel A).

Apoptosis-induced mRNA degradation acts on all mRNAs analyzed independent of their intrinsic half-lives and the transcriptional status of the gene (constitutively expressed or inducible genes) and affects mRNA coding for different types of proteins (structural, proapoptotic, cell type-specific, etc.). mRNA degradation precedes DNA fragmentation and correlates with the appearance of phosphatidylserine in the outer cell membrane. mRNAs with different half-lives show similar apoptosis-induced degradation kinetics, indicating that mRNA degradation is an active process induced by apoptosis rather than the consequence of a transcriptional arrest.

2. Apoptosis-induced mRNA degradation is controlled by a distinct signaling pathway
Pretreatment of cells (A20 or Jurkat) with the general caspase inhibitor Z-VAD-fmk was able to block not only the appearance of annexin V-positive cells, DNA degradation, and PARP processing, but also cytoplasmic mRNA degradation induced by direct CD95 cross-linking. When apoptosis was induced for 6 h with anti-CD95 mAb and the caspase inhibitor Z-VAD-fmk was added at different times, conditions were found in which DNA degradation, annexin V binding, and PARP degradation were inhibited whereas mRNA degradation still occurred (Fig. 2 ). These data also demonstrate that though the increase in PARP processing, annexin V-positive cells, and cells in sub-G₀/G₁ is progressive, mRNA degradation is an all-or-none effect (Fig. 2) .

View larger version (46K): [in this window] [in a new window]   Figure 2. Kinetics of Z-VAD-fmk apoptosis inhibition in A20 cells allows discrimination between mRNA degradation and other apoptotic markers. Kinetic effects of Z-VAD-fmk were analyzed on A20 cells treated for 6 h with anti-Fas monoclonal antibody, where the inhibitor was added at different times ranging from 1 h before to 1.5 h after the addition of anti-Fas antibodies. As controls, untreated cells were 3% annexin V-positive, 1% in sub-G0/G1, and PARP processing was undetectable; anti-Fas-treated cells were 73% annexin V-positive, 59% of the cells were in the sub-G0/G1 phase of the cycle, the vast majority of PARP was processed, and 84% of mRNA was degraded. A) Methylene blue staining of the RNA samples, hybridization with the specific murine IA mRNA, and detection of PARP processing by Western blot. B) Quantification of annexin V-positive cells, cells in sub G0/G1, and IA mRNA degradation.

Similar results were obtained using JHM12 cells and were corroborated by experiments overexpressing the baculoviral caspase inhibitor p35. These data allowed us to dissect apoptosis-induced mRNA degradation from other apoptotic hallmarks (annexin V, DNA, and PARP degradation) and strongly suggest that apoptosis-induced mRNA degradation is controlled by a distinct signaling pathway.

3. Apoptosis-induced mRNA degradation occurs in vivo as an early apoptotic event
Apoptosis-induced mRNA degradation occurs in in vivo models such as negative selection in the thymus, which in F5-TCR mice can be mimicked by antigenic peptide injection, which leads to the massive apoptosis of immature thymocytes (CD4⁺CD8⁺). When no reduction in cell numbers occurred (90 min after peptide injection), PARP processing and DNA fragmentation were detected in thymocytes from peptide-injected animals but not from PBS-injected control animals. Specific mRNA degradation was demonstrated in thymocytes from antigen-injected animals concomitant with the appearance of the other apoptotic markers. The fraction of degraded transcripts was higher than the corresponding fraction of fragmented DNA.

CONCLUSION

Taken together, our data demonstrate that apoptosis-induced mRNA degradation is an early event triggered by different apoptotic signals, occurring not only in in vitro models but also in vivo. It is not restricted to genes with a specific function, since besides 28S rRNA, it affects mRNA coding for proteins implicated in a variety of functions, including cell type-specific functions such as HLA-I, IA, TCRß, and CD69, structural functions such as ß-actin, or control of cell survival, such as BAX.

Apoptosis-induced mRNA degradation is an active process that induces a similar decay kinetics (t_1/2 between 1.5 and 3 h) of mRNAs with very different intrinsic half-lives (from 20 min to >10 h). Kinetic experiments using general caspase inhibitors strongly suggest that the signaling pathways triggered by the death stimuli diverge into one pathway leading to caspase activation (PARP processing, DNA degradation, and annexin V binding) and another leading to mRNA degradation. In addition, our data show that extensive mRNA degradation, although apparently a general early apoptotic event, is not sufficient to induce cell death per se.

The molecular mechanisms implicated in apoptosis-induced mRNA degradation are not known (Fig. 3 ). Nonetheless, it seems that the apoptotic stimuli leads to activation of an RNase dependent on an upstream apical caspase (inhibited by Z-VAD-fmk, but not p35, and unable to cleave PARP) in a similar manner as CAD activation. Conversely, activation of the RNase may be independent of caspase activation but still inhibited by Z-VAD-fmk.

View larger version (91K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing the intracellular effects of apoptotic signals. An external death signal is transduced to the cytoplasm leading to activation of caspases and proteolysis of caspase substrates, including PARP, ICAD, etc. As a consequence, phosphatidyl serine is exposed in the outer layer of the cell membrane and CAD can translocate to the nucleus and cleave the genomic DNA. The initiation factor eIF4G is another well-known substrate of caspases; its cleavage inhibits translation of cap-dependent transcripts. It is also known that 28S rRNA is cleaved by RNAse L during apoptosis, and the data presented here demonstrate that mRNA degradation is induced during apoptosis by a mechanism that may be independent of caspases. The data suggest that an initial effect of apoptosis signals is a general blockade of translation that would prevent any cellular repair mechanism for which newly synthesized proteins are required.

The nature of ribonuclease(s) involved remains to be elucidated. RNase L has been implicated in the degradation of 28S rRNA during apoptosis; it is ubiquitous and was described as an interferon-inducible enzyme. Since, RNase L^-/- mice show defective apoptosis in thymus and spleen as well as in in vitro thymocyte- and fibroblast-induced apoptosis, it is possible to speculate that this RNase may be involved in apoptosis-induced mRNA degradation.

Thus, apoptosis induces degradation of the cytoplasmic mRNAs but also degradation of 28S ribosomic RNA (rRNA) and cleavage of translation initiation factors (eIF4G I, eIF4GII), which suggests that the cell blocks protein synthesis by different means, which may be a prerequisite to reach the "point of no return" in the cell death program. Degradation of cellular mRNA is a general early apoptotic event that may be considered a hallmark of apoptosis. In addition to the implications in the physiology of apoptosis, this has clear implications in the field of mRNA turnover, since it demonstrates for the first time a generalized change in mRNA stability induced by an external stimuli and will have to be taken into account in experiments aiming to identify genes involved in the cell death process.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0392fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0392fje

² Present address: Escuela Técnica Superior de Ingenieros Agrónomos, Univ. Politécnica de Madrid, Madrid, Spain

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