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. Mitkevich
1
, Nickolai A. Tchurikov
1
, Pavel V. Zelenikhin
2
, Irina Yu. Petrushanko
1
,
Alexander A. Makarov
1
and Olga N. Ilinskaya
1,2
1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
2 Department of Microbiology, Kazan State University, Kazan, Russia
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 alter-
native 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 pipi-
ens 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 amylolique-
faciens RNase (barnase) cleaves a wide range of
noncoding tRNAs and rRNAs, thus significantly
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: ;

(Received 28 July 2009, revised 8 October
2009, accepted 2 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. Cleav-
age 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 sensi-
tive to the cytotoxic effects of binase. The RNA level was lowered by 44%
in HEK cells transfected with the hSK4 gene of the Ca
2+

-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. Kit-
transformed cells with binase-induced RNA decrease retained viability if
the interleukin-dependent proliferation pathway was activated. Using quan-
titative RT-PCR with RNA samples isolated from the binase-treated HE-
KhSK4 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-depen-
dent processes of cell proliferation and apoptosis.
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
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 RNA-
induced 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 deter-
mined by Polyakov et al. [23]. We have shown that
binase selectively inhibits the growth of cells, express-
ing 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 cellu-
lar 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-trans-
formed FDC-P1iR1171 cells, whose RNA level is
decreased by binase, does not change if the interleukin
(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 expres-
sion 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 signifi-
cantly 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, visu-
ally selected on the basis of characteristic morphology
and high granulation had a higher fluorescence inten-
sity 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.
AB
C
a
a
b
bc
ca b
Fig. 1. Visualization of cellular RNA by stain-
ing 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 l
M binase for 48 h.
V. A. Mitkevich et al. Binase affects cellular RNA
FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 187
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 HE-
KhSK4, 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 sensi-
tive 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 fluo-
rescence 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 cyto-
solic fractions of FDC-P1 and FDC-P1iR1171 cells
was measured with a poly(I) substrate stable to pyrimi-

dine-specific eukaryotic RNases [24], in order to deter-
mine 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 treat-
ment with binase led to similar effects for both cell
lines: within 24 h after addition of binase, the hydro-
lysis rate in both the nuclear and the cytosolic fractions
increased two-fold to six-fold (Fig. 4A,D). The differ-
ence 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 FDC-
P1iR1171 cells, IL led to an increase in the mean
RNA level in the population by 20% (Fig. 5, +IL col-
umns). 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
Table 1. 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 5 ± 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%.
Cell line
Viability of
binase-treated cells (%)
Apoptotic cells (%)

RNA content of
binase-treated cells (%)Early apoptotic cells Late apoptotic cells
FDC-P1iR1171 67 ± 7
a
36 ± 4 3 ± 1 80 ± 4
HEKhSK4 85 ± 3 8 ± 2 10 ± 2 56 ± 3
FDC-P1 96 ± 2 20 ± 6 2 ± 1 95 ± 5
a
According to [17].
AB
Fig. 2. Binase lowers the amount of RNA in
the cells. Intracellular RNA contents in sub-
populations of viable (A) and apoptotic (B)
HEKhSK4 and FDC-P1iR1171 cells untreated
and treated with binase (+Bi) (48 h, 40 l
M),
detected by staining with SYTO RNASelect.
Binase affects cellular RNA V. A. Mitkevich et al.
188 FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 com-
pared with those grown on medium without binase
(Fig. 5, +IL+Bi and +IL columns).
Hydrolysis of the hSK4 mRNA by RNases in vitro
Figure 6 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 full-
length 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 frag-
ments, with only a small amount being found in the
19–31 nucleotide range (Fig. 6A,B), and the
32
P-
labeled 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
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 bin-

ase, 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 treat-
ment, 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
Fig. 3. Binase reduces the proportions of HEKhSK4 and FDC-
P1iR1171 cells with high RNA contents. The ratio of the subpopula-
tions 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 l
M). Subpopulations were selected
according to the cytometric distribution of fluorescence intensity.
A
B
C
D
E
F
Fig. 4. Binase increases the poly(I) hydrolysis rate in cells. Cleav-
age 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 l
M binase for 24 h (A, D), 48 h (B,
E), and 72 h (C, F).
V. A. Mitkevich et al. Binase affects cellular RNA
FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 189

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 cel-
lular 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 abil-
ity 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 leuke-
mia cells, FDC-P1iR1171 transgenic myeloid progeni-
tor 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 apoptosis-
inducing action of binase on FDC-P1iR1171 and HE-
KhSK4 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 apop-
tosis process by the RNase. Additional evidence for
this is the retention of viability of binase-treated FDC-
P1iR1171 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. There-
fore, 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
Fig. 5. 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 l
M binase
(+Bi) for 72 h in the presence (+IL) or absence ()IL) of IL. The via-
bility 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.
Binase affects cellular RNA V. A. Mitkevich et al.
190 FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS
the high fluorescence levels are caused by the dye bind-
ing 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 proba-
bly in the late stages of apoptosis, and do 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 sensi-
tive 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 con-
cluded that binase uses noncoding RNA as a substrate,
as only approximately 5% of the genome output con-
sists of protein-coding mRNAs [31]. This is in agreement
with information about the massive cleavage of tRNA
A

B
Fig. 6. Digestions of the hSK4
32
P-labeled
transcripts by RNases. (A) Cleavage prod-
ucts 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 sepa-
rated in a sequencing gel, and autoradio-
graphed using X-ray film. Lane 1: untreated
RNA. Lanes 2–4: products of ssRNA diges-
tion with 5K RNase Sa, RNase Sa, and bin-
ase, 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 inteference-
related regulation mechanisms.
V. A. Mitkevich et al. Binase affects cellular RNA
FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 191
and rRNA in SKOV-3 cells by a close analog of binase,
i.e. barnase, which was visualized using gel electrophore-
sis 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 homolo-
gous and their biochemical properties are almost identi-
cal [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 cat-
alytic 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
do 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].
Recently, the effect of onconase on the dsRNA was
demonstrated [33]. We have shown that the single-
strand-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) do not allow us to elucidate any special fea-
tures 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 non-
toxic [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
We observed upregulation of the K
Ca
channels and the
apoptotic p53 genes and downregulation of the antia-
poptotic 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 synthe-
sis 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
K
Ca
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 K
Ca

channels
[18]. This indicates that an exogenous binase, after
penetrating into the cells, can affect the regulatory
mechanisms of RNA-dependent processes of transcrip-
tion 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
Fig. 7. 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 l
M binase for
24 h and 48 h.
Binase affects cellular RNA V. A. Mitkevich et al.
192 FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 proa-
poptotic 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 dis-
ruption 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 l m, except for those used for RT-PCR
analysis (see below), which were treated with 15 lm binase,
because when they were treated with 40 l m binase it was
impossible to extract enough of the cellular RNA for analy-
sis. 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).
Cell cultures
Four different cell lines were used. Human embryonic kid-
ney cells transfected with the DNA encoding the human
small conductance Ca
2+
-activated K
+
channel type hSK4
(HEKhSK4) and immortalized human kidney epithelial
embryo cells (IHKE) were obtained from the Rudolf-
Buchheim Institute of Pharmacology (Giessen, Germany).
Normal myeloid progenitor cells (FDC-P1) and transgenic
myeloid progenitor cells expressing the activated kit onco-
gene (FDC-P1iR1171) were obtained from the Heinrich-
Pette Institute of Experimental Virology and Immunology
(Hamburg, Germany). The IHKE cells were cultured
according to [42], and the HEKhSK4, FDC-P1 and FDC-

P1iR1171 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 min at 37 °C. The fluorescence intensi-
ties 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 pre-
loaded with 5 lm SYTO RNASelect (excitation at 490 nm;
emission at 530 nm) for 30 min at 37 °C for RNA detec-
tion. Cell viability was assessed by adding propidium iodide
(Molecular Probes) at a final concentration of 10 lgÆmL
)1
to the cell suspension for 1–2 min before measurements.
Apoptosis was verified with annexin V–fluorescein isothio-
cyanate (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 min 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 min was
observed on a Cary Eclips fluorimeter (Varian, Palo Alto,
CA, USA).
V. A. Mitkevich et al. Binase affects cellular RNA
FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS 193
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 ⁄ P
i
(Sigma-Aldrich). Then, lysis buffer (10 mm Hepes,
10 mm KCl, 2 mm MgCl
2
, 0.5% NP-40 in double-distilled
H
2
O, pH 7.9) supplemented with Protease Inhibitor Cock-
tail (PIC; Roche Diagnostics, Mannheim, Germany) was
added, and the suspension was centrifuged at 1500 g for
5 min at 4 °C. The supernatant was used as the cytosolic
fraction, and the pellet was resuspended in a low-salt solu-
tion (20 mm Hepes, 2.5 l L of 25% glycerol, 2 mm MgCl
2
,

0.1 mm KCl, 1 mm EDTA, pH 7.9) supplemented with
PIC, and again centrifuged at 1500 g for 5 min at 4 °C.
The pellet was resuspended in Tris buffer (10 mm Tris ⁄ HCl,
3mm MgCl
2
,2mm 2-mercaptoethanol, 5 mm CH
3
COONa,
0.5% SDS, 0.5 mm CaCl
2
, 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 1 min
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
32
P-labeled sense transcript, a
cDNA clone (pcDNA3; Invitrogen, Carlsbad, CA, USA)
was used. The
32
P-labeled sense and the unlabeled antisense
transcripts were synthesized in vitro using T7 RNA poly-
merase on DNA templates digested with NotIorBamHI,
respectively. The reactions were performed in 20 lLofa

solution containing 1 lg of DNA, 40 mm Tris ⁄ HCl (pH
7.5), 6 mm MgCl
2
,2mm spermidine, 10 mm NaCl, 10 mm
dithiothreitol, 1 unitÆlL
)1
RNasin (where one unit is
defined as the amount of RNasin Ribonuclease inhibitor
required to inhibit the activity of 5 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 1 lm [
32
P]UTP[aP] (6000 CiÆmmol
)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
32
P-labeled sense RNA in 10 lL of solution con-
taining 100 mm NaCl, 10 mm Tris ⁄ HCl (pH 7.5), 2 mm
MgCl
2

, and 0.5 units of RNasin. Annealing was performed
for 5 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
32
P-labeled sense
RNA with a 20-fold excess of unlabeled antisense RNA
(data not shown). Self-annealing of
32
P-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 2 lL of annealed samples and 3 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 min at 37 ° C. Then, 7 lL of dyes
containing formamide and 2 mm EDTA were added. The
mixture was maintained at 90 °C for 3 min, and used for
electrophoresis.
Fractionation
For fractionation, 0.2-mm-thick denaturing 12% polyacryl-
amide gel containing 8.3 m urea was used. Two-microliter
samples were loaded onto each lane. The
32
P-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 phos-
phoimager (Packard Instruments, Meriden, CT, USA), and

exposed to X-ray film.
RT-PCR analysis
RNA was isolated from about 150 · 10
3
HEKhSK4 cells
using the Trizol reagent (Invitrogen). Samples were treated
with DNase using a DNA-free kit (Ambion), and approxi-
mately 2 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 with-
out 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
Binase affects cellular RNA V. A. Mitkevich et al.
194 FEBS Journal 277 (2010) 186–196 ª 2009 The Authors Journal compilation ª 2009 FEBS
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% Nu-
Sieve agarose gels, and the separation data were evaluated
using quantity one quantitation software (Bio-Rad, Her-
cules, 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 spe-
cific 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 differ-
ent 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 Biol-

ogy 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
President of the Russian Federation for young scientists
(MK-162.2009.4).
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