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• W1994107946 endingPage "24807" @default.
• W1994107946 startingPage "24799" @default.
• W1994107946 abstract "We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell. We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell. caspase-activated DNase ribonucleoprotein particle small nuclear Apoptosis is a form of cell death characterized by distinct morphological and biochemical alterations. Morphologically, apoptotic cells are characterized by chromatin condensation, cell shrinkage, fragmentation of the nucleus, and partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies) (1Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1101) Google Scholar). During the last 5 years, many of the molecules that participate in the biochemical pathway mediating apoptosis have been identified. A major role in this pathway is played by caspases, cysteine proteases with aspartic acid substrate specificity (2Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2124) Google Scholar). Proteins cleaved by caspases appear to be structural proteins essential for maintaining nuclear and cytoplasmic architecture and enzymes essential for repair of damaged cell components (reviewed in Ref. 3Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4084) Google Scholar). A prominent nuclear event during apoptosis is internucleosomal cleavage of DNA, recognized as a “DNA ladder” on conventional agarose gel electrophoresis (4Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4129) Google Scholar). The endonuclease activity responsible for apoptotic degradation of chromosomal DNA has recently been identified (5Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2785) Google Scholar). The activity depends on two interacting proteins, one of which contains the endonuclease activity (caspase-activated deoxyribonuclease (CAD)1), which is retained in the cytoplasm in an inactive form due to its association with the second protein (inhibitor of CAD). Caspase activation in apoptotic cells leads to cleavage of the inhibitor of CAD, thereby releasing active CAD resulting in DNA fragmentation in the nuclei (5Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2785) Google Scholar, 6Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1409) Google Scholar). Much less is known about cleavage and degradation of RNA in apoptotic cells. An increased rate of mRNA turnover has been suggested (7Mondino A. Jenkins M.K. J. Biol. Chem. 1995; 270: 26593-26601Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Owens G.P. Cohen J.J. Cancer Metastasis Rev. 1992; 11: 149-156Crossref PubMed Scopus (36) Google Scholar) as well as mitochondrial 16 S ribosomal RNA degradation (9Crawford D.R. Lauzon R.J. Wang Y. Mazurkiewicz J.E. Schools G.P. Davies K.J. Free Radical Biol. Med. 1997; 22: 1295-1300Crossref PubMed Scopus (47) Google Scholar), but no nuclease associated with specific RNA cleavage has been described. Although an increasing number of protein components of ribonucleoprotein particles (RNPs) have been reported to be modified during apoptosis, such as the U1-70K protein, which is a component of the U1 snRNP (10Casciola-Rosen L.A. Miller D.K. Anhalt G.J. Rosen A. J. Biol. Chem. 1994; 269: 30757-30760Abstract Full Text PDF PubMed Google Scholar), the 72-kDa component of the signal recognition particle (11Utz P.J. Hottelet M. Le T.M. Kim S.J. Geiger M.E. Van Venrooij W.J. Anderson P. J. Biol. Chem. 1998; 273: 35362-35370Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and the La protein, which is associated with several RNPs including the Ro RNPs, 2S. A. Rutjes, P. J. Utz, A. van der Heijden, C. Broekhuis, W. J. van Venrooij, and G. J. M. Pruijn, submitted for publication.2S. A. Rutjes, P. J. Utz, A. van der Heijden, C. Broekhuis, W. J. van Venrooij, and G. J. M. Pruijn, submitted for publication.no data have been published on the fate of the RNA components of these particles during apoptosis. Therefore, we decided to examine the effects of apoptosis on the cytoplasmic Y RNAs and 7SL RNA, the RNA components of the Ro RNPs and the signal recognition particle, respectively. Ro RNPs are a class of small cytoplasmic RNA-protein complexes of unknown function, which are present in cells of all species studied to date (reviewed in Ref. 12Pruijn G.J.M. Simons F.H.M. Van Venrooij W.J. Eur. J. Cell Biol. 1997; 74: 123-132PubMed Google Scholar). In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY1, hY3, hY4, and hY5 (13Hendrick J.P. Wolin S.L. Rinke J. Lerner M.R. Steitz J.A. Mol. Cell. Biol. 1981; 1: 1138-1149Crossref PubMed Scopus (308) Google Scholar). All four human Y RNAs have been sequenced (14Kato N. Hoshino H. Harada F. Biochem. Biophys. Res. Commun. 1982; 108: 363-370Crossref PubMed Scopus (39) Google Scholar, 15O'Brien C.A. Harley J.B. EMBO J. 1990; 9: 3683-3689Crossref PubMed Scopus (52) Google Scholar, 16Wolin S.L. Steitz J.A. Cell. 1983; 32: 735-744Abstract Full Text PDF PubMed Scopus (120) Google Scholar) and found to consist of 112, 101, 93, and 84 nucleotides, respectively, although some heterogeneity at their 3′-ends has been observed. The Y RNAs, which are transcribed by RNA polymerase III (13Hendrick J.P. Wolin S.L. Rinke J. Lerner M.R. Steitz J.A. Mol. Cell. Biol. 1981; 1: 1138-1149Crossref PubMed Scopus (308) Google Scholar), are characterized by a conserved stem structure formed by extensive base pairing between the evolutionarily conserved 5′- and 3′-ends. In addition to Y RNAs, Ro RNPs contain at least two different proteins: the La protein and the 60-kDa Ro protein (Ro60), whereas the association of a third protein, the 52-kDa Ro protein (Ro52), is still a matter of debate (17Ben Chetrit E. Chan E.K. Sullivan K.F. Tan E.M. J. Exp. Med. 1988; 167: 1560-1571Crossref PubMed Scopus (335) Google Scholar, 18Boire G. Gendron M. Monast N. Bastin B. Menard H.A. Clin. Exp. Immunol. 1995; 100: 489-498Crossref PubMed Scopus (75) Google Scholar, 19Peek R. Pruijn G.J.M. Van Venrooij W.J. J. Immunol. 1994; 153: 4321-4329PubMed Google Scholar, 20Slobbe R.L. Pluk W. Van Venrooij W.J. Pruijn G.J.M. J. Mol. Biol. 1992; 227: 361-366Crossref PubMed Scopus (90) Google Scholar). The La protein binds to the oligouridylate stretch at the 3′-end of the Y RNAs, whereas Ro60 interacts with the most highly conserved part of the stem structure (21Pruijn G.J.M. Slobbe R.L. Van Venrooij W.J. Nucleic Acids Res. 1991; 19: 5173-5180Crossref PubMed Scopus (104) Google Scholar, 22Wolin S.L. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1996-2000Crossref PubMed Scopus (295) Google Scholar). In this study, we observed an extensive, rapid, and selective nucleolytic degradation of small cytoplasmic RNAs, the Y RNAs, during apoptosis. This phenomenon was observed upon exposure of the cells to multiple apoptotic stimuli, and yRNA degradation appeared to be inhibited by the apoptosis inhibitors Bcl-2 and zinc, as well as by the caspase inhibitors Ac-YVAD-CMK, Z-DEVD-FMK, and Z-IETD-FMK. The results of co-immunoprecipitation experiments and size determination of the apoptotic Y RNA degradation products suggest that the most divergent regions of the Y RNAs are degraded and that the association of Ro60 with the conserved regions is not disrupted, whereas the association with La is partially lost. Jurkat cells, with Bcl-2 (Jurkat/Bcl-2) or without Bcl-2 (Jurkat/Neo) overexpression, kindly provided by Dr. J. Reed (the Burnham Institute, La Jolla, CA) (23Torigoe T. Millan J.A. Takayama S. Taichman R. Miyashita T. Reed J.C. Cancer Res. 1994; 54: 4851-4854PubMed Google Scholar), were grown in RPMI (Life Technologies, Inc.) medium supplemented with 10% heat-inactivated fetal calf serum, 200 μg/ml G418 (Life Technologies, Inc.), 1 μm β-mercaptoethanol, 1 mm sodium pyruvate, and penicillin and streptomycin. Mouse WR19L cells expressing human Fas (24Cuppen E. Nagata S. Wieringa B. Hendriks W. J. Biol. Chem. 1997; 272: 30215-30220Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) were grown in RPMI (Life Technologies, Inc.) medium supplemented with 10% heat-inactivated fetal calf serum, 200 μg/ml G418 (Life Technologies, Inc.), 1 μm β-mercaptoethanol, 1 mm sodium pyruvate, and penicillin and streptomycin. HeLa cells and HEp-2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and penicillin and streptomycin. Cells were cultured in 5% CO2at 37 °C. Jurkat/Bcl-2 and Jurkat/Neo cells and WR19L cells expressing human Fas (24Cuppen E. Nagata S. Wieringa B. Hendriks W. J. Biol. Chem. 1997; 272: 30215-30220Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) were treated with an anti-Fas monoclonal antibody 7C11, a kind gift of Dr. M. Robertson (Indiana University, Bloomington, IN), and cells were incubated at 37 °C for the indicated time periods prior to harvesting. HeLa cells were treated with 10 μm actinomycin D and HEp-2 cells with 10 μg/ml anisomycin. Total RNA was isolated by Trizol RNA reagent (Life Technologies, Inc.), according to the instructions of the manufacturer. In parallel, cell extracts were prepared by lysis in Nonidet P-40 lysis buffer (25 mm Tris, pH 7.5, 100 mm KCl, 0.25 mm dithioerythritol, 10 mm MgCl2, 1% Nonidet P-40, protease inhibitor mixture from Roche Molecular Biochemicals) for 30 min on ice. After centrifugation for 15 min at 12,000 × g, supernatants were analyzed by Western blotting. Monolayer cells were trypsinized, washed with phosphate-buffered saline, and treated as above. For experiments utilizing caspase inhibitors, Jurkat cells were cultured in the presence of either 2% Me2SO, 2 mmZnSO4, 2 or 20 μm Ac-YVAD-CMK (caspase-1 inhibitor 2, Calbiochem), 2 or 20 μm Z-DEVD-FMK (caspase-3 inhibitor 2, Calbiochem), 2 or 20 μmZ-IETD-FMK (caspase-8 inhibitor 2, Calbiochem) and 2 or 20 μm Z-LEHD-FMK (caspase-9 inhibitor 1, Calbiochem). Subsequently, apoptosis was induced by the addition of anti-Fas monoclonal antibody followed by harvesting after incubations as indicated and lysis as described above. RNA was size-fractionated on 10% denaturing polyacrylamide gels and transferred to Hybond N+filters by electroblotting at 3 V/cm in 0.025 m phosphate, pH 6.5, for 2 h. Hybridizations were performed overnight at 65 °C in 6× SSC, 5× Denhardt's solution, and 100 μg/ml sheared, denatured herring sperm DNA with a mixture of 32P-labeled antisense RNA transcripts of the four hY RNAs, antisense 7SL RNA (25Strub K. Moss J. Walter P. Mol. Cell. Biol. 1991; 11: 3949-3959Crossref PubMed Scopus (83) Google Scholar), kindly provided by Dr. K. Strub (University of Geneva, Switzerland), or antisense U1 snRNA. Following hybridization, filters were washed twice at 65 °C in 0.2× SSC, 0.1% SDS and were subjected to autoradiography. Transcription of antisense hY1, hY3, hY4, hY5, 7SL, and U1 RNA was mainly performed as described (26Scherly D. Boelens W. Van Venrooij W.J. Dathan N.A. Hamm J. Mattaj I.W. EMBO J. 1989; 8: 4163-4170Crossref PubMed Scopus (254) Google Scholar). To obtain antisense RNAs, pTZ19-hY1, hY3, hY4, hY5, SP64–7SL, and pGEM-U1 RNA were linearized with Eco RI. In vitro transcription was performed with T7 RNA polymerase (antisense hY1, hY3, hY4, and hY5 RNA) or SP6 RNA polymerase (antisense U1 and 7SL RNA). Transcription and 3′-end labeling of tRNAHis was performed as described previously (27Brouwer R. Vree Egberts W. Jongen P.H. Van Engelen B.G.M. Van Venrooij W.J. Arthritis Rheum. 1998; 41: 1428-1437Crossref PubMed Scopus (31) Google Scholar). Cell extracts were fractionated by SDS-polyacrylamide gel electrophoresis (10%) and blotted onto nitrocellulose filter. After blocking the filters in wash buffer (5% skim milk, phosphate-buffered saline, 0.1% Nonidet P-40) for 1 h at room temperature, filters were incubated with patient serum H42 (anti-U1-70K) at a dilution of 1:5000 in wash buffer for 1 h at room temperature. After washing three times for 15 min, binding of antibodies was visualized by incubation with peroxidase-conjugated rabbit anti-human antibodies (Dako) followed by chemiluminescence detection. Jurkat/Bcl-2 and Jurkat/Neo cells were incubated at a density of 2 × 106 cells/ml in labeling medium (RPMI without phosphate (ICN), 2 mmGlutaMAX (Life Technologies, Inc.), 5% dialyzed fetal calf serum 1 mm sodium pyruvate, 10 mm Hepes (pH 7.4), and penicillin and streptomycin). 32P-labeled orthophosphate was added at a concentration of 33 μCi/ml. After incubating the cells at 37 °C for 18 h, an equal volume of RPMI (Life Technologies, Inc.) medium supplemented with 10% heat-inactivated fetal calf serum, 200 μg/ml G418 (Life Technologies, Inc.), 1 μmβ-mercaptoethanol, 1 mm sodium pyruvate, 10 mm Hepes (pH 7.4), and penicillin and streptomycin was added. Cells were treated with an anti-Fas monoclonal antibody 7C11 and incubated at 37 °C. RNA was isolated either immediately or after incubation for 1, 2, 3, 4, 6, or 8 h. For protein analysis, radiolabeled cells were solubilized in Nonidet P-40 lysis buffer at the indicated time points. Cell lysates were incubated on ice for 30 min, followed by centrifugation for 15 min at 4 °C at 12,000 ×g. To immunoprecipitate either unlabeled or radiolabeled RNA, protein A-agarose beads were incubated with rabbit anti-mouse antibodies (Dako) for at least 1 h in IPP500 (10 mm Tris-HCl (pH 7.5), 500 mm NaCl, 0.05% Nonidet P-40). After washing three times with IPP500, the beads were incubated with an anti-Ro60 monoclonal antibody (2G10), an anti-La monoclonal antibody (SW5), or an anti-U1-A monoclonal antibody (9A9) by rotation for at least 1 h in IPP500. Incubation was followed by washing twice with IPP500 and twice with TKED (10 mm Tris-HCl (pH 8.0), 100 mm KCl, 1 mm dithioerythritol, 1 mm EDTA, 0.05% Nonidet P-40). After rotating the coated beads with the extracts in TKED for 2 h at 4 °C, the beads were washed three times with TKED. RNA was isolated by phenol/chloroform (1:1) extraction and was precipitated by adding 4 volumes of ethanol and analyzed by 10% denaturing polyacrylamide gel electrophoresis. Radiolabeled RNA was subjected to autoradiography, whereas unlabeled RNA was analyzed by Northern blot hybridization. To study the effects of apoptosis on the hY RNAs and 7SL RNA, we used two stably transfected Jurkat cell lines, one overexpressing the apoptosis inhibitor Bcl-2 (Jurkat/Bcl-2) and the second a transfection vector control line (Jurkat/Neo). To induce apoptosis, the cells were treated with a monoclonal antibody reactive with Fas (7C11). Previous studies have demonstrated that these antibodies very effectively induce apoptosis in Jurkat cells (11Utz P.J. Hottelet M. Le T.M. Kim S.J. Geiger M.E. Van Venrooij W.J. Anderson P. J. Biol. Chem. 1998; 273: 35362-35370Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 28Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar).2 Cells were harvested either immediately or at the indicated time points after anti-Fas addition. Total RNA was isolated from cell extracts and analyzed by Northern blot hybridization using 32P-labeled antisense hY1, hY3, hY4, hY5, and 7SL RNA probes. The induction of apoptosis was monitored by the analysis of U1-70K protein cleavage, which leads to the appearance of a characteristic 40-kDa product. Cleavage of U1-70K, which is one of the prototypical proteins known to be cleaved during apoptosis (10Casciola-Rosen L.A. Miller D.K. Anhalt G.J. Rosen A. J. Biol. Chem. 1994; 269: 30757-30760Abstract Full Text PDF PubMed Google Scholar), was visualized by immunoblotting of cell extracts using a patient serum reactive with the U1-70K protein. Analysis of the hY RNAs revealed that during early stages of apoptosis, these RNAs were efficiently degraded in anti-Fas-treated Jurkat/Neo cells (Fig.1 A). Degradation products were already detectable 1.5 h after anti-Fas addition (lane 13), whereas a gradual decrease in the amount of intact hY RNAs was evident, with the majority of the hY RNAs being degraded within 4 h after anti-Fas addition (compare lane 18 with lane 11). Although all four hY RNAs were degraded upon induction of apoptosis, slight differences were observed in the rate of degradation. The rate of degradation appeared to be related to the size of the hY RNA; hY1 was degraded most quickly. In contrast, no degradation of 7SL RNA was observed in these cells (Fig.1 A). The selectivity of degradation of hY RNAs in apoptotic cells was further substantiated by the lack of detectable degradation of several other small RNAs, including U snRNAs, tRNAs, and 5 S rRNA (data not shown). Degradation of hY RNAs was inhibited in Jurkat cells overexpressing Bcl-2, because a slight decrease of the amount of hY RNAs was only detectable 6 h after anti-Fas addition, whereas degradation products did not appear during the first 3 h (lanes 1–10). The delayed degradation of hY RNAs in Jurkat/Bcl-2 cells in comparison with the Jurkat/Neo cells reflected the different efficiencies of apoptosis induction in these cells, which was monitored by cleavage of the U1-70K protein (Fig. 1 B) and by flow cytometry of annexin-V stained cells (results not shown).2The kinetics of hY RNA degradation appeared to be very similar to that of U1-70K cleavage, which is known to be mediated by caspase-3, suggesting that hY RNA degradation might also be dependent on caspase activation. To determine whether degradation of hY RNAs also occurs when apoptosis is triggered by other stimuli, and in cells derived from other species, we analyzed apoptotic cell extracts derived from human HeLa cells treated with actinomycin D (10 μm) (data not shown) or HEp-2 cells treated with anisomycin (10 μg/ml). Also, apoptotic cell extracts, derived from mouse WR19L cells expressing human Fas (24Cuppen E. Nagata S. Wieringa B. Hendriks W. J. Biol. Chem. 1997; 272: 30215-30220Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and treated with anti-Fas monoclonal antibody, were analyzed. Total RNA was isolated and analysis of Y RNAs by Northern blot hybridization revealed that Y RNA degradation was observed in all cells tested (Fig.1 C). Induction of apoptosis in all cells was confirmed by cleavage of the U1-70K protein. Although the normal turnover rate of hY RNAs is known to be relatively low, a reduction of the hY RNA levels in apoptotic cells might in principle be due to either inhibition of RNA polymerase III transcription or to an increased rate of hY RNA degradation. To exclude the possibility that the reduction in hY RNA levels was caused by abrogation of their synthesis and to investigate the association of the Ro RNP with hY RNAs in apoptotic cells, RNA in Jurkat/Bcl-2 and Jurkat/Neo cells was radiolabeled by culturing the cells in the presence of 32P-orthophosphate for 18 h. After labeling, apoptosis was induced by anti-Fas addition in complete medium, i.e. in the presence of an excess of unlabeled phosphate. Cells were lysed either immediately or after incubation for the indicated time periods, and induction of apoptosis was monitored by cleavage of the U1-70K protein (data not shown). The results for the Jurkat/Bcl-2 cells illustrate the low turnover rate of hY RNAs (Fig.2, lanes 5–11). Even at 8 h after replacement of radiolabeled phosphate with unlabeled phosphate, little or no decrease of radiolabeled hY RNAs was observed, which is indicative of the relatively long half-life of these molecules. This result confirms that the observed decrease in hY RNAs levels during apoptosis is due to a strongly increased degradation rate rather than the abrogation of hY RNA synthesis. Because an immunoprecipitation step was required to isolate the low abundance hY RNAs from the total pool of radiolabeled RNAs, this experiment also provided information on the Ro RNP association of radiolabeled hY RNAs. Immunoprecipitation was performed with an anti-Ro60 monoclonal antibody (2G10), and co-precipitated RNAs were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The results in Fig. 2 show that co-immunoprecipitation of hY RNAs from apoptotic Jurkat/Neo cell extracts decreased during the first hours after anti-Fas addition and was hardly detectable at the 4 h time point (lanes 10–14). The decrease in hY RNA precipitation by anti-Ro60 antibodies is likely to be indeed caused by hY RNA degradation rather than by disruption of the interaction between hY RNAs and Ro60, because the analysis of Ro60 from apoptotic cells by Western blotting did not reveal detectable changes, such as proteolytic cleavage (Ref. 28Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar and data not shown). Moreover, the RNA binding capacity of Ro60 was not abolished in apoptotic Jurkat cells, as demonstrated by the co-precipitation of hY RNA degradation products (see below). It should also be noted that the disappearance of full-length hY RNAs isolated by immunoprecipitation from radiolabeled cell extracts resembles the decrease of hY RNA signals obtained by Northern blot analysis of total RNA (Fig. 1). Taken together, these results demonstrate that the disappearance of hY RNAs during apoptosis is indeed due to degradation and that hY RNAs remain in association with the Ro RNP in apoptotic cells until or even after the degradation process has been initiated. The differences in RNA ranging in size approximately from 5 to 5.8 S rRNA between total radiolabeled RNA isolated either immediately after anti-Fas addition (Fig. 2, lanes 1 and 3) or following 8 h of incubation (lanes 2 and 4) might be due to apoptotic degradation of ribosomal RNA and/or mRNA (7Mondino A. Jenkins M.K. J. Biol. Chem. 1995; 270: 26593-26601Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Owens G.P. Cohen J.J. Cancer Metastasis Rev. 1992; 11: 149-156Crossref PubMed Scopus (36) Google Scholar, 9Crawford D.R. Lauzon R.J. Wang Y. Mazurkiewicz J.E. Schools G.P. Davies K.J. Free Radical Biol. Med. 1997; 22: 1295-1300Crossref PubMed Scopus (47) Google Scholar), resulting in higher background signals. Note that due to the low abundance of radiolabeled hY RNAs, relatively high background signals of much more abundant RNAs, such as 5 and 5.8 S rRNA (Fig. 2, lanes 1 and 3), were observed among the immunoprecipitated RNAs (lanes 5–14). Caspases are not only involved in the activation of apoptotic proteases; also, caspase-dependent activation of a deoxyribonuclease has recently been reported (5Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2785) Google Scholar). To study the role of caspases in the activation of the nuclease activity responsible for hY RNA degradation during apoptosis, Jurkat cells were cultured in the presence of several caspase inhibitors, including zinc sulfate (29Takahashi A. Alnemri E.S. Lazebnik Y.A. Fernandes-Alnemri T. Litwack G. Moir R.D. Goldman R.D. Poirier G.G. Kaufmann S.H. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8395-8400Crossref PubMed Scopus (469) Google Scholar), the caspase-1 inhibitor Ac-YVAD-CMK, the caspase-3 inhibitor Z-DEVD-FMK, the caspase-8 inhibitor Z-IETD-FMK, or the caspase-9 inhibitor Z-LEHD-FMK for 1 h prior to and during anti-Fas treatment. Cells were harvested either immediately or 4 or 8 h after anti-Fas addition. Total RNA was isolated and analyzed by Northern blotting using hY RNA probes and a 7SL RNA probe as a control. Fig. 3 A demonstrates that hY RNA degradation was completely inhibited in Jurkat cells cultured in the presence of zinc sulfate (Fig. 3 A, lanes 4–6). hY RNA degradation was also clearly inhibited by the addition of Ac-YVAD-CMK, Z-DEVD-FMK and Z-IETD-FMK (Fig. 3 B, lanes 3–8) in comparison with the control incubation with 2% Me2SO (Fig.3 B, lane 2). In contrast, the caspase-9 inhibitor Z-LEHD-FMK only poorly affected hYRNA degradation (Fig. 3 B, lanes 9–10). As expected, the addition of these inhibitors had no effect on 7SL RNA signals (Fig. 3, lower panels). As a control for the inhibitory activity of the tetrapeptide inhibitors, the cell extracts were also analyzed for U1-70K cleavage, which is known to be sensitive to Ac-DEVD-CHO (10Casciola-Rosen L.A. Miller D.K. Anhalt G.J. Rosen A. J. Biol. Chem. 1994; 269: 30757-30760Abstract Full Text PDF PubMed Google Scholar, 28Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar, 30Casciola Rosen L. Nicholson D.W. Chong T. Rowan K.R. Thornberry N.A. Miller D.K. Rosen A. J. Exp. Med. 1996; 183: 1957-1964Crossref PubMed Scopus (575) Google Scholar). Cleavage of the U1-70K protein was indeed inhibited by ZnSO4 and the caspase-1, caspase-3, and caspase-8 inhibitors and to a lesser extent by the caspase-9 inhibitor (data not shown). These results demonstrate that the apoptotic degradation of hY RNAs is dependent on caspase activation. The results described above indicate that hY RNAs remain associated with the Ro60 protein until or possibly even during the degradation process. Therefore, it was possible that at least some of the degradation products were still bound by either the Ro60 or the La protein, the two proteins that are directly bound to the hY RNAs in Ro RNP complexes. To investigate the potential interaction of these proteins with the apoptotic degradation products hY RNAs, immunoprecipitation experiments were performed with monoclonal anti-Ro60 (2G10) and anti-La (SW5) antibodies. Cell extracts were prepared at various time points after the addition of anti-Fas antibody and RNA was analyzed by Northern blot hybridization either directly isolated from cell extracts or following immunoprecipitation. Fig. 4 A shows RNA isolated from cell extracts, corresponding with 10% of the cell extracts used for immunoprecipitation. As is shown in Fig. 4, B and C, both the full-length hY RNAs and at least part of the degradation products were co-immunoprecipitated with anti-Ro60 (Fig.4 B) and anti-La (Fig. 4 C) antibodies. This strongly suggests that both proteins remain associated with the hY RNAs during the nucleolytic process and thus with the respective binding site containing degradation products of the hY RNAs. The anti-Ro60 antibody co-precipitated the degradation products much more efficiently than the anti-La antibody, which might indicate that although both antibodies seem to co-precipitate the same set of degradation products, the La binding site might be partially lost. A control immunoprecipitation was performed with a monoclonal antibody (9A9) to the U1A protein, a protein specifically associated with the U1 snRNP. As expected, U1 snRNA was not co-precipitated with anti-Ro60 and anti-La antibodies, and no hY RNAs were co-precipitated with the anti-U1A antibodies (Fig. 4 D). In contrast, U1 snRNA was efficiently precipitated by the anti-U1A antibodies, substantiating the specificity of the immunoprecipitations. To determine the length of the apoptotic degradation products of the hY RNAs, 32P-labeled Jurkat/Neo cell extracts were used to isolate h" @default.
• W1994107946 created "2016-06-24" @default.
• W1994107946 creator A5010973711 @default.
• W1994107946 creator A5027596376 @default.
• W1994107946 creator A5056082573 @default.
• W1994107946 creator A5081889079 @default.
• W1994107946 creator A5090309778 @default.
• W1994107946 date "1999-08-01" @default.
• W1994107946 modified "2023-10-14" @default.
• W1994107946 title "Rapid Nucleolytic Degradation of the Small Cytoplasmic Y RNAs during Apoptosis" @default.
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