Báo cáo khoa học: Binase cleaves cellular noncoding RNAs and affects coding mRNAs potx

Binase cleaves cellular noncoding RNAs and affects coding mRNAs Vladimir A Mitkevich1, Nickolai A Tchurikov1, Pavel V Zelenikhin2, Irina Yu Petrushanko1, Alexander A Makarov1 and Olga N Ilinskaya1,2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia Department of Microbiology, Kazan State University, Kazan, Russia Keywords apoptosis; cellular RNA degradation; cytotoxicity; gene expression; RNase Correspondence A A Makarov or O N Ilinskaya, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia Fax: +7 499 1351405 Tel: +7 499 1354095 E-mail: aamakarov@eimb.ru; olga.ilinskaya@ksu.ru (Received 28 July 2009, revised October 2009, accepted November 2009) doi:10.1111/j.1742-4658.2009.07471.x Bacterial RNases are promising tools for the development of anticancer drugs Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca2+-activated potassium channels (HEKhSK4) and by 20% in kit-transformed myeloid progenitor FDC-P1iR1171 cells The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells However, the binase-induced RNA decrease did not correlate with apoptosis Kittransformed cells with binase-induced RNA decrease retained viability if the interleukin-dependent proliferation pathway was activated Using quantitative RT-PCR with RNA samples isolated from the binase-treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl-2 gene in vivo was reduced about two-fold In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5-fold and 4.3-fold, respectively These results show that binase is a regulator of RNA-dependent processes of cell proliferation and apoptosis Introduction RNA is an active player in oncogenesis [1–3] One of the signs of neoplastic transformation is the enhanced accumulation of rRNA and tRNA and broadly altered expression of microRNAs (miRNAs), all of which are categorized as noncoding RNA RNases therefore possess therapeutic possibilities for cancer treatment, as RNA damage caused by RNases could be an alternative to standard DNA-damaging chemotherapeutics [4–8] The ribonucleolytic activity of exogenously applied RNases is essential for their cytotoxicity [4,5,9] Degradation of tRNA by onconase (Rana pipiens RNase) [10,11], 28S rRNA by Aspergillus RNase a-sarcin [12] and 16S rRNA by colicin E3 (Escherichia coli RNase) [13] have been shown Bacillus amyloliquefaciens RNase (barnase) cleaves a wide range of noncoding tRNAs and rRNAs, thus significantly Abbreviations IL, interleukin; miRNA, microRNA; PIC, Protease Inhibitor Cocktail; RT, reverse transcriptase 186 FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS V A Mitkevich et al Binase affects cellular RNA decreasing the total amount of intracellular RNA in ovarian carcinoma SKOV-3 cells [14] Zhao et al [15] have shown that one of the targets of onconase is short interfering RNA, probably within the RNAinduced silencing complex Recent data on the cytotoxic effects of microbial RNases, such as Bacillus intermedius RNase (binase) [16–18], barnase [14], Streptomyces aureofaciens RNase Sa3 [19] and the 5K cationic mutant of RNase Sa [18,20,21], suggest that RNases of the T1 superfamily have promise as a basis for developing new antitumor drugs Binase is a highly cationic guanyl-specific RNase that catalyzes RNA cleavage without the need for metal ions and cofactors Cloning and sequencing of the binase gene have been reported [22], and a three˚ dimensional structure at 1.65 A resolution was determined by Polyakov et al [23] We have shown that binase selectively inhibits the growth of cells, expressing ras [16] and kit [17] oncogenes In this work, we investigated what effects binase has on the total amount of intracellular RNA and on the expression of proapoptotic and antiapoptotic mRNAs We provide evidence that the level of noncoding cellular RNA decreases in cells that are sensitive to the cytotoxic effects of binase This effect is most easily observed in a subpopulation of apoptotic cells However, the binase-induced RNA decrease does not correlate with apoptosis The viability of the kit-transformed FDC-P1iR1171 cells, whose RNA level is decreased by binase, does not change if the interleukin A a (IL)-dependent proliferation pathway is activated We have found in vivo that binase affects the quantity of proapoptotic and antiapoptotic mRNAs, as expression of the p53 and hSK4 genes was increased and expression of the bcl-2 gene was reduced Results Visualization of the cellular RNA The most intensive fluorescence in IHKE cells, after RNA dye treatment, was found in the prenuclear part of the endoplasmic reticulum (Fig 1Aa–c) Among IHKE cells treated with binase and those not treated with binase, there were no individual cells that significantly differed from the others by their fluorescence However, cells treated with binase tended to show a reduction in the radius of the fluorescing zone around the nucleus (Fig 1B), thus demonstrating a decrease in the amount of RNA in this zone Apoptotic cells from FDC-P1iR1171 culture, visually selected on the basis of characteristic morphology and high granulation had a higher fluorescence intensity than viable cells in samples both treated and untreated with binase (Fig 1C) At the same time, among the apoptotic cells, individuals with practically no RNA were detected (Fig 1Ca) The amount of such cells in the total population did not exceed 5% In order to obtain quantitative data on the RNA level in the cells before and after binase treatment, flow cytometry was used b C a B a c b b c Fig Visualization of cellular RNA by staining with SYTO RNASelect (A) RNA in IHKE cells: (a) without staining; (b) merged image; (c) with staining (B) IHKE cells untreated (a) and treated (b) with binase, (C) RNA in binase-treated FDC-P1iR1171 cells: (a) without staining; (b) merged image; (c) with staining White arrows (Ca) show individual apoptotic cells without RNA Cells were treated with 40 lM binase for 48 h FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 187 Binase affects cellular RNA V A Mitkevich et al Table Viability, apoptosis and RNA content of binase-treated cells (48 h, 40 lM) The viability of control cells grown without RNases was set to 100% The total amount of binase-treated cells was set as 100% The amounts of apoptotic cells in untreated cultures were ± 3% for HEKhSK4 cells and 18 ± 7% for FDC-P1 and FDCP1iR1171 cells The RNA levels of cells grown without RNases were set as equal to 100% Apoptotic cells (%) Cell line Viability of binase-treated cells (%) Early apoptotic cells Late apoptotic cells RNA content of binase-treated cells (%) FDC-P1iR1171 HEKhSK4 FDC-P1 67 ± 7a 85 ± 96 ± 36 ± 8±2 20 ± 3±1 10 ± 2±1 80 ± 56 ± 95 ± a According to [17] Binase lowers the amount of RNA in the cells Binase lowered cell viability and increased the numbers of apoptotic cells in lines FDC-P1iR1171 and HEKhSK4, but did not affect cells from line FDC-P1 (Table 1) The apoptosis-inducing effect of binase was accompanied by a lowered level of RNA (Table 1) The decline in RNA levels was not proportional to the intensity of the cytotoxic effect: in the cells most sensitive to binase, from line FDC-P1iR1171, the total drop in the amount of RNA after binase treatment was up to 20% in comparison with untreated cells, whereas in line HEKhSK4 it was up to 44% (Table 1) The most significant decrease in RNA levels in both cell lines was seen in the subpopulation of apoptotic cells (Fig 2) Also, binase reduced the proportions of HEKhSK4 and FDC-P1iR1171 cells with high fluorescence intensities (Fig 3), whereas for the FDC-P1 cells this effect was not shown Binase increases the poly(I) hydrolysis rate in cells The ribonucleolytic activity of the nuclear and cytosolic fractions of FDC-P1 and FDC-P1iR1171 cells A was measured with a poly(I) substrate stable to pyrimidine-specific eukaryotic RNases [24], in order to determine the activity of the guanyl-specific binase after its penetration into the cells Without binase treatment, the rate of hydrolysis of poly(I) in these fractions was less than 0.2–0.4 relative units per cell, whereas treatment with binase led to similar effects for both cell lines: within 24 h after addition of binase, the hydrolysis rate in both the nuclear and the cytosolic fractions increased two-fold to six-fold (Fig 4A,D) The difference in the rate of hydrolysis of poly(I) between the treated and native cells remained the same for 48 h (Fig 4B,E), and then disappeared after 72 h of enzyme action (Fig 4C,F) IL removes the apoptotic, but not the RNA-decreasing, effect of binase When added to the cultivation medium of FDCP1iR1171 cells, IL led to an increase in the mean RNA level in the population by 20% (Fig 5, +IL columns) In cells grown on medium without IL, in 72 h binase increased the number of apoptotic cells by 30% and lowered the viability of the culture by 70%, while decreasing the total amount of RNA by only 13% as B Fig Binase lowers the amount of RNA in the cells Intracellular RNA contents in subpopulations of viable (A) and apoptotic (B) HEKhSK4 and FDC-P1iR1171 cells untreated and treated with binase (+Bi) (48 h, 40 lM), detected by staining with SYTO RNASelect 188 FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS V A Mitkevich et al Binase affects cellular RNA A D B E C F Fig Binase reduces the proportions of HEKhSK4 and FDCP1iR1171 cells with high RNA contents The ratio of the subpopulations with low (blank sectors) and high (filled sectors) RNA content in HEKhSK4, FDC-P1 and FDC-P1iR1171 cells untreated and treated with binase (+Bi) (72 h, 40 lM) Subpopulations were selected according to the cytometric distribution of fluorescence intensity compared with cells untreated with binase (Fig 5, )IL+Bi and )IL columns) It was found that IL did not decrease the catalytic activity of binase in vitro (data not shown) In the presence of IL, binase did not affect the viability of the FDC-P1iR1171 cells and their transition into apoptosis, despite the fact that the amount of RNA in them decreased by 23% as compared with those grown on medium without binase (Fig 5, +IL+Bi and +IL columns) Hydrolysis of the hSK4 mRNA by RNases in vitro Figure shows patterns of the RNA fragments observed after the digestion of a single-stranded and a double-stranded hSK4 mRNA sample with binase, the nontoxic RNase Sa, and its highly cytotoxic cationic mutant 5K RNase Sa [20], used for comparison All of the RNases actively digested the ssRNA, and the fulllength RNA product was not observed: RNase Sa and 5K RNase Sa generated RNA fragments in the range from 20 to 200 nucleotides, whereas binase digested the same sample more deeply into very short fragments, with only a small amount being found in the 19–31 nucleotide range (Fig 6A,B), and the 32Plabeled dsRNA was only partially cleaved by excessive amounts of RNase Sa, 5K RNase Sa, and binase The distribution of the dsRNA hydrolysis products was shifted towards longer fragments (Fig 6A,B) Binase was clearly much less active on the dsRNA substrate than RNase Sa and 5K RNase Sa The patterns of RNA fragments generated from dsRNA by RNase Sa and 5K RNase Sa are practically the same About 15% of the labeled products from ssRNA and 6% from dsRNA were found in the 19–35 nucleotide Fig Binase increases the poly(I) hydrolysis rate in cells Cleavage of poly(I) by nuclear and cytosolic fractions of FDC-P1 cells (A–C) and FDC-P1iR1171 cells (D–F) untreated (blank columns) and treated (filled columns) with 40 lM binase for 24 h (A, D), 48 h (B, E), and 72 h (C, F) region for both 5K RNase Sa and RNase Sa As for binase, less than 1% of the label was revealed in this region for both ssRNA and dsRNA samples Binase affects the quantity of proapoptotic and antiapoptotic mRNAs in vivo To test whether the expression of some genes that are involved in the control of apoptosis is affected by binase, we selected three genes – the proapoptotic p53 tumor suppressor [25], the antiapoptotic bcl-2 [26], and the ion channel gene hSK4, with a dual function in apoptosis [27] Using quantitative RT-PCR on RNA samples isolated from the HEKhSK4 cells treated by binase, we observed that, after 24 h of binase treatment, the amount of bcl-2 mRNA was slightly reduced, and that after 48 h it had dropped about two-fold (Fig 7) In contrast, the p53 steady-state FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 189 Binase affects cellular RNA V A Mitkevich et al Fig IL abolishes the apoptotic, but not the RNA-decreasing, effect of binase Viability (blank columns), the amount of apoptotic cells (black columns) and cellular RNA content (shaded columns) for FDC-P1iR1171 cells untreated and treated with 40 lM binase (+Bi) for 72 h in the presence (+IL) or absence ()IL) of IL The viability and cellular RNA contents of cells grown without binase and IL were taken as 100% The amount of apoptotic cells is expressed as a proportion of the total number of cells of each type expression was maintained during the first 24 h of treatment, and increased 1.5-fold after 48 h (Fig 7) Expression of the hSK4 gene increased about two-fold after 24 h of treatment with binase, and then rose more than four-fold as compared with the steady-state expression These data showed that binase does not simply lead to the degradation of some RNAs, but affects the regulation of gene transcription Discussion The binase-induced RNA decrease does not correlate with apoptosis There are no doubts that cytotoxic RNases affect cellular RNA They cleave a wide range of substrates: tRNA by onconase [10,11], 28S rRNA by a-sarcin [12], 16S rRNA by colicin E3 [13], and tRNA and all types of rRNA by barnase [11] It is assumed that the cytotoxicity of the onconase is associated with its ability to cleave miRNAs [15] and to degrade RNA to form products similar to short interfering RNAs [28] Binase induces apoptosis in HEK and HEKhSK4 embryonic kidney cells [18], K562 myelogenous leuke190 mia cells, FDC-P1iR1171 transgenic myeloid progenitor cells expressing activated kit oncogene [17], and NIH3T3 ras-expressing fibroblasts [16], but does not affect normal fibroblasts [16] and normal FDC-P1 myeloid progenitor cells [17] The lack of the specific target (kit) for binase in the latter cells does not induce their death even after internalization of the enzyme, as shown by the increase in the rate of hydrolysis of poly(I) after 24 h of binase treatment (Fig 4A,D) This increase precedes the binase-induced apoptosis, which develops within 48 h after treatment only in kit-transformed cells (Table 1) and disappears in 72 h, probably due to the action of intracellular proteases (Fig 4C,F) Here, we have demonstrated that the apoptosisinducing action of binase on FDC-P1iR1171 and HEKhSK4 cells is accompanied by a decrease in the total amount of RNA (Table 1) and in the number of cells with a high RNA content (Fig 3), whereas in the case of FDC-P1 cells, which are insensitive to binase, these effects are not observed However, even though the decrease in RNA in HEKhSK4 cells is more than two times greater than in FDC-P1iR1171 cells, the latter are more susceptible to apoptosis (Table 1) Also, onconase induces apoptosis in mitogen-stimulated lymphocytes, but does not affect the total RNA content [29] Thus, the reduction in the RNA level is not directly connected with the induction of the apoptosis process by the RNase Additional evidence for this is the retention of viability of binase-treated FDCP1iR1171 cells in the presence of IL, despite the fact that the RNA content in these cells is 23% lower than without the binase (Fig 5) Addition of IL leads to the activation of its own antiapoptosis and proliferation pathways, regardless of the Kit-dependent signaling pathways [30], thus canceling the binase-induced growth depression of the kit-transformed cells Therefore, RNA breakdown by itself does not cause cell death The effect of binase-induced reduction of the RNA content is greatest in the subpopulation of apoptotic cells Under binase action, the total RNA content in the apoptotic subpopulation of HEKhSK4 cells decreases much more severely than in the viable subpopulation (Fig 2) Among FDC-P1iR1171 cells, binase lowers the RNA levels only in apoptotic cells Apoptotic FDC-P1iR1171 cells demonstrated higher fluorescence levels of the RNA-bound dye than viable cells, even though, physiologically, they could not have had greater RNA amounts (Fig 1Cb) It is possible that FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS V A Mitkevich et al Binase affects cellular RNA A B Fig Digestions of the hSK4 32P-labeled transcripts by RNases (A) Cleavage products of the hSK4 mRNA fragment digested in vitro with 5K RNase Sa, RNase Sa, and binase The original ssRNA and the same RNA after annealing with the excess of unlabeled antisense RNA were digested with the RNases The samples were separated in a sequencing gel, and autoradiographed using X-ray film Lane 1: untreated RNA Lanes 2–4: products of ssRNA digestion with 5K RNase Sa, RNase Sa, and binase, respectively Lanes 5–7: products of dsRNA digestion with 5K RNase Sa, RNase Sa, and binase, respectively (B) Enlarged fragment of the same gel M: RNA marker, with lengths of fragments in nucleotides The brackets show the RNA fragments corresponding in size (19–31 nucleotides) to small RNAs involved in RNA inteferencerelated regulation mechanisms the high fluorescence levels are caused by the dye binding to the rRNA, which is freed from the proteins, as their expression in apoptotic cells stops The possibility that the increase in fluorescence intensity was caused by the emergence of additional dye-binding sites on the RNA during hydrolysis was ruled out, because RNA fluorescence during experiments in vitro with the SYTO RNASelect dye steadily decreased with time under binase action (data not shown) Even though the number of apoptotic cells increases under binase action (Table 1), their fluorescence decreases (Fig 2B), and some of them are not even stained by SYTO RNASelect (Fig 1Ca, arrows) These cells are probably in the late stages of apoptosis, and not contain macromolecular RNA Such cells are also found in insignificant numbers in the populations without binase treatment The death of cell lines that are sensitive to binase treatment is more significant for the FDC-P1iR1171 line, in which only 3% of late apopto- tic cells are left, than for the HEKhSK4 line, where about 10% of such cells remain (Table 1) Even though, under binase action, the level of RNA in apoptotic cells of the HEKhSK4 line dropped more severely than in cells from line FDC-P1iR1171 (Fig 2B), they remained in the culture for much longer periods of time This once more confirms that there is no straight correlation between lowered RNA levels and cell death Noncoding RNAs are obligatory substrates for binase Considering the significant drop in the RNA level of transformed cells (20–44%; Table 1), it can be concluded that binase uses noncoding RNA as a substrate, as only approximately 5% of the genome output consists of protein-coding mRNAs [31] This is in agreement with information about the massive cleavage of tRNA FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 191 Binase affects cellular RNA V A Mitkevich et al Recently, the effect of onconase on the dsRNA was demonstrated [33] We have shown that the singlestrand-preferring RNase binase [23] cleaves the dsRNA (Fig 6) Degradation of the dsRNA by binase occurs through the same mechanism as described for RNase Sa and a number of other single-strand-preferring RNases [34,35] The patterns of the RNA hydrolysis products obtained with microbial RNases under investigation (Fig 6) not allow us to elucidate any special features relating to the cytotoxicity of the RNases Thus, the nontoxic RNase Sa and its cytotoxic 5K mutant [20] have the same effect on the dsRNA (Fig 6), whereas binase, which is less active towards the dsRNA, is cytotoxic [20] Anionic RNase Sa is nontoxic [20], whereas the cationic RNases – binase and 5K RNase Sa, which have essentially different patterns of RNA hydrolysis products – are both cytotoxic [20] Binase affects gene expression Fig Binase affects the quantity of proapoptotic and antiapoptotic mRNAs The mRNA expression levels of genes hSK4, bcl-2 and p53 in HEKhSK4 cells untreated and treated with 15 lM binase for 24 h and 48 h and rRNA in SKOV-3 cells by a close analog of binase, i.e barnase, which was visualized using gel electrophoresis in polyacrylamide gel [14] In this work, it was shown that the relative abundance of tRNA and 5.8S, 5S, 18S and 28S rRNA after barnase treatment was significantly decreased As the two RNases are very highly homologous and their biochemical properties are almost identical [32], one can assume that binase should have practically the same effect on RNA as barnase and should cleave both tRNA and rRNA in cells The decrease of the RNA content is caused by the catalytic effect of the enzyme in the cells Binase treatment of FDC-P1iR1171 and FDC-P1 myeloid progenitor cells leads, within 24 h, to a significant increase of the RNase hydrolytic activity on poly(I), in both the cytosolic and the nuclear fractions (Fig 4A) These results agree with the data on the decrease in the radius of the fluorescent zone formed by RNA staining in IHKE cells (Fig 1B) After 48 h, when the poly(I) hydrolysis rate returns to the background level (Fig 4B,C), the apoptotic action of binase becomes apparent (Table 1) Features of the RNA hydrolysis process in vitro not allow toxic and nontoxic RNases to be distinguished The effects on the dsRNA are especially significant in the case of antitumor activities of RNases [6] 192 We observed upregulation of the KCa channels and the apoptotic p53 genes and downregulation of the antiapoptotic bcl-2 gene under binase action (Fig 7) These changes are typical for apoptosis [36], and indicate that the mRNA of antiapoptotic genes can, to some extent, contribute to the overall binase-induced decrease in the cellular RNA levels Activation of p53 mRNA synthesis is not in agreement with the overall decrease in the RNA levels, and thus indicated a regulatory role for binase Besides this, activation of expression of the KCa channel gene, which is important for shrinkage of the apoptotic cell, develops only at the mRNA level We have determined that binase, like 5K RNase Sa, depresses the functional activity of the KCa channels [18] This indicates that an exogenous binase, after penetrating into the cells, can affect the regulatory mechanisms of RNA-dependent processes of transcription and translation Degradation of the available RNA by exogenous RNases puts the cellular RNA levels out of balance and disrupts the regulatory processes, thus leading to the induction of apoptosis It is possible to assume that the effect on gene expression will take place if the length of the duplex fragments generated by bacterial RNases corresponds to that of small, noncoding RNAs involved in RNA regulation mechanisms The possibility of such duplex formation was shown in vitro by experiments on digestion of the hSK4 gene mRNA by the RNases used in this study (Fig 6) Similarly, it has been demonstrated that onconase cleaves tRNA in vitro, yielding fragments 5–40 nucleotides in length [11] The possibility also cannot be excluded that FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS V A Mitkevich et al RNases may degrade small cellular RNAs, which are repressors of gene translation This deserved special attention, as it is known that some miRNAs from the miR17–92 cluster are amplified and overexpressed in human cancers, thus blocking the translation of proapoptotic genes [37] Selective degradation of these miRNAs could potentially prevent the repression of apoptosis, and therefore be exploited for anticancer chemotherapy [3] A similar mechanism was suggested for onconase [15] Recently, it has been reported that small RNA duplexes generated by hydrolysis with E coli RNase III mediate effective RNA interference in mammalian cells [38] Thus, by targeting different regulatory RNAs with cytotoxic RNases, it might be possible to regulate the expression of certain genes Conclusions The choice between life and death of a cell subjected to exogenous RNases depends on the characteristic pattern of hydrolysis products of the cellular RNA, which reflects the results of complicated interactions between molecular determinants of RNases on one side [5,7] and the cellular targets on the other [16,17,20] The results of this investigation show that binase reduces the amount of RNA in sensitive cells, but this decrease by itself is not fatal, as it is the disruption of RNA-dependent regulatory processes that causes cell death The development of an approach based on information about the RNA regulation network and the creation of more selective cationic microbial RNases targeting specific RNAs could be promising Experimental procedures Enzymes Binase (12.3 kDa) was isolated from the culture fluid of E coli BL21 carrying plasmid pGEMGX1 ⁄ ent ⁄ Bi as a homogeneous protein The enzyme purification was carried out by a procedure described in [32] Binase was assayed for catalytic activity towards synthetic substrates [32] and yeast RNA [39] The cells were treated with binase at a concentration of 40 lm, except for those used for RT-PCR analysis (see below), which were treated with 15 lm binase, because when they were treated with 40 lm binase it was impossible to extract enough of the cellular RNA for analysis RNase Sa and 5K RNase Sa (D1K, D17K, E41K, D25K, and E74K) [40,41] were gifts from J M Scholtz and C N Pace (Texas A&M University System Health Science Center, College Station, TX, USA) Binase affects cellular RNA Cell cultures Four different cell lines were used Human embryonic kidney cells transfected with the DNA encoding the human small conductance Ca2+-activated K+ channel type hSK4 (HEKhSK4) and immortalized human kidney epithelial embryo cells (IHKE) were obtained from the RudolfBuchheim Institute of Pharmacology (Giessen, Germany) Normal myeloid progenitor cells (FDC-P1) and transgenic myeloid progenitor cells expressing the activated kit oncogene (FDC-P1iR1171) were obtained from the HeinrichPette Institute of Experimental Virology and Immunology (Hamburg, Germany) The IHKE cells were cultured according to [42], and the HEKhSK4, FDC-P1 and FDCP1iR1171 cells were cultured as described previously [17,18] RNA visualization by fluorescence microscopy RNA in the IHKE and FDC-P1iR1171 cells was stained with 2.5 lm SYTO RNASelect (Molecular Probes, Eugene, OR, USA) for 30 at 37 °C The fluorescence intensities of intracellular RNA after staining and washing off of the extra dye were analyzed using saved images obtained with a Leica DM 6000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and Leica fw4000 software Determination of RNA level, viability and apoptosis by flow cytometry HEKhSK4, FDC-P1 and FDC-P1iR1171 cells were preloaded with lm SYTO RNASelect (excitation at 490 nm; emission at 530 nm) for 30 at 37 °C for RNA detection Cell viability was assessed by adding propidium iodide (Molecular Probes) at a final concentration of 10 lgỈmL)1 to the cell suspension for 1–2 before measurements Apoptosis was verified with annexin V–fluorescein isothiocyanate (Molecular Probes) [43] and propidium iodide [44] double staining All measurements were performed on a Beckman Coulter Epix XL4 flow cytometer (Fullerton, CA, USA) In order to show that degradation of the RNA does not lead to an increase in the fluorescence intensity values, the fluorescence of a solution of RNA, stained with SYTO RNASelect, in the presence of binase was measured SYTO RNASelect (0.5 lm) was added to 11 lm yeast RNA (Sigma-Aldrich, St Louis, MO, USA) in Tris buffer (10 mm Tris ⁄ HCl, 140 mm NaCl, pH 7.0) After 10 of RNA staining, 1.8 · 10)8 m binase was added, and at 530 nm a decrease in the fluorescence intensity by 80% in 15 was observed on a Cary Eclips fluorimeter (Varian, Palo Alto, CA, USA) FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 193 Binase affects cellular RNA V A Mitkevich et al Extraction of the cytosolic and nuclear fractions of the cell, and determination of the RNase activity in these fractions FDC-P1iR1171 and FDC-P1 cells were washed in ice-cold NaCl ⁄ Pi (Sigma-Aldrich) Then, lysis buffer (10 mm Hepes, 10 mm KCl, mm MgCl2, 0.5% NP-40 in double-distilled H2O, pH 7.9) supplemented with Protease Inhibitor Cocktail (PIC; Roche Diagnostics, Mannheim, Germany) was added, and the suspension was centrifuged at 1500 g for at °C The supernatant was used as the cytosolic fraction, and the pellet was resuspended in a low-salt solution (20 mm Hepes, 2.5 lL of 25% glycerol, mm MgCl2, 0.1 mm KCl, mm EDTA, pH 7.9) supplemented with PIC, and again centrifuged at 1500 g for at °C The pellet was resuspended in Tris buffer (10 mm Tris ⁄ HCl, mm MgCl2, mm 2-mercaptoethanol, mm CH3COONa, 0.5% SDS, 0.5 mm CaCl2, PIC, pH 7.6), and treated as described in [45] in order to obtain the nuclear fraction Cleavage of poly(I) (Sigma-Aldrich) by the nuclear and cytosolic fractions was measured as described by Yakovlev et al [46] RNase activity was expressed in relative units, which show the increase in absorption at 248 nm in divided by the number of cells used to obtain the cytosolic and nuclear fractions The number of cells was calculated using a Leica DMI 4000B light microscope RNA synthesis For synthesis of the hSK4 32P-labeled sense transcript, a cDNA clone (pcDNA3; Invitrogen, Carlsbad, CA, USA) was used The 32P-labeled sense and the unlabeled antisense transcripts were synthesized in vitro using T7 RNA polymerase on DNA templates digested with NotI or BamHI, respectively The reactions were performed in 20 lL of a solution containing lg of DNA, 40 mm Tris ⁄ HCl (pH 7.5), mm MgCl2, mm spermidine, 10 mm NaCl, 10 mm dithiothreitol, unitỈlL)1 RNasin (where one unit is defined as the amount of RNasin Ribonuclease inhibitor required to inhibit the activity of ng of ribonuclease A by 50%; as defined by the manufacturer, Promega, Madison, WI, USA), ATP, GTP, CTP (500 lm each), 10 lm UTP, and lm [32P]UTP[aP] (6000 CiỈmmol)1, ‘Phosphor-center’ of the Russian Academy of Sciences, Moscow, Russia), and 20 units of the T7 RNA polymerase The conditions used favor the synthesis of full-length RNA products Annealing To ensure the annealing of full-length molecules, RNA preparations were gel-purified in 4% acrylamide gel, using a mirVana miRNA detection kit for elution (Ambion, Austin, TX, USA) About 0.6 ng of antisense RNA was mixed with 0.03 ng of 32P-labeled sense RNA in 10 lL of solution con- 194 taining 100 mm NaCl, 10 mm Tris ⁄ HCl (pH 7.5), mm MgCl2, and 0.5 units of RNasin Annealing was performed for h under mineral oil, using a temperature shift from 50 to 35 °C Annealing was tested by gel-shift assay, which demonstrated the complete annealing of 32P-labeled sense RNA with a 20-fold excess of unlabeled antisense RNA (data not shown) Self-annealing of 32P-labeled ssense RNA under the same conditions was performed as a control RNase treatment The RNase digestions were performed in 20 lL-well plates containing lL of annealed samples and lL of annealing mixture Up to 0.1 lg of RNase (5K RNase Sa, RNase Sa, and binase) were added to the wells, and the incubation was performed for 30 at 37 °C Then, lL of dyes containing formamide and mm EDTA were added The mixture was maintained at 90 °C for min, and used for electrophoresis Fractionation For fractionation, 0.2-mm-thick denaturing 12% polyacrylamide gel containing 8.3 m urea was used Two-microliter samples were loaded onto each lane The 32P-labeled RNA markers were synthesized on pGEM1 DNA digested with EcoRI or SmaI After electrophoresis, the gel was washed with 10% acetic acid, dried, scanned using a Cyclone phosphoimager (Packard Instruments, Meriden, CT, USA), and exposed to X-ray film RT-PCR analysis RNA was isolated from about 150 · 103 HEKhSK4 cells using the Trizol reagent (Invitrogen) Samples were treated with DNase using a DNA-free kit (Ambion), and approximately lg of RNA Specific primers and Moloney murine leukemia virus reverse transcriptase (RT) (Promega) were used for synthesis of cDNAs corresponding to the hSK4 mRNA, p53 mRNA and bcl-2 mRNA, according to the manufacturer’s instructions Each PCR was performed with the cDNA template (RT+) and the same RNA probe without addition of RT The number of PCR cycles varied from 28 to 37 Primers for RT-PCR were selected according to the primer selection tool program (http://biotools umassmed.edu/) The primers for cDNA synthesis were as follows: 5¢TGGAAGCTGCCTCGGCCCCAGGGC-3¢ (for hSK4 mRNA), 5¢-GGCCCTTCTGTCTTGAACATGAG-3¢ (for p53 mRNA), and 5¢-AATATTAACTAGACAGACAAGG AAA-3¢ (for bcl-2 mRNA) For quantitative PCR with cDNA corresponding to sense transcripts of hSK4, bcl-2, and p53, the following primers were used: 5¢-GAAGCCTG GATGTTCTACAAACATA-3¢ and 5¢-AAGCAGCTCAGT FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS V A Mitkevich et al CAGGGCATCC-3¢, 5¢-CTAATGGTGGCCAACTGGAG ACT-3¢ and 5¢-GTTTTGTTTATTATACCTTCTTAAGTT TT-3¢, and 5¢-AGACCGGCGCACAGAGGAAGAGAA-3¢ and 5¢-CTTTTTGGACTTCAGGTGGCTGG-3¢, respectively Conditions for linear PCR for each set of primers were selected in the preliminary experiments using Mastercycler personal (Eppendorf, Hamburg, Germany) The final PCR products were separated in mixed 1% agarose ⁄ 2% NuSieve agarose gels, and the separation data were evaluated using quantity one quantitation software (Bio-Rad, Hercules, CA, USA) Statistical analysis of the fractionated DNA fragments obtained in five independent experiments was performed with origin software The identity of the amplified DNA fragments was confirmed by sequencing The RT-PCR data were normalized to ribosomal human 5.8S RNA as follows Two-microliter aliquots from the final 40 lL cDNA probes synthesized using different specific primers on total RNA preparations (see above) were used for new cDNA synthesis with the 5.8S RNA specific primer 5¢-GCTCAGACAGGCGTAGCCCCGGGA-3¢ One microliter of the sample was then taken for PCR using 5.8S gene-specific primers 5¢-CGGTGGATCACTCGGC TCGT-3¢ and 5¢-GCCGCAAGTGCGTTCGAAGTG-3¢ The data from RNA preparations corresponding to different constructs were normalized using quantity one Acknowledgements We thank M Scholtz and N Pace for presenting us with RNase Sa and its 5K mutant We thank C Stocking for donating the FDC-P1 and FDC-P1iR1171 cells We thank A Koschinski for preparing the IHKE cells This work was supported by the Molecular and Cellular Biology Program of the Russian Academy of Sciences, by the RFBR (grant 07-04-01051), by the Russian Federal Programs (contracts 02.512.11.2198, 02.512.12.2014, 2.1.1 ⁄ 920, and 02.740.11.0391), and by a Grant of the 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[20] Binase affects gene expression Fig Binase affects the quantity of proapoptotic and antiapoptotic mRNAs The mRNA expression levels of genes hSK4, bcl-2 and p53 in HEKhSK4 cells untreated and. .. 2–4: products of ssRNA digestion with 5K RNase Sa, RNase Sa, and binase, respectively Lanes 5–7: products of dsRNA digestion with 5K RNase Sa, RNase Sa, and binase, respectively (B) Enlarged fragment... microbial RNases, such as Bacillus intermedius RNase (binase) [16–18], barnase [14], Streptomyces aureofaciens RNase Sa3 [19] and the 5K cationic mutant of RNase Sa [18,20,21], suggest that RNases