ABSTRACT
The
pac1
+ gene of the fission yeast
Schizosaccharomyces pombe
is essential for viability and its overexpression induces sterility and
suppresses mutations in the
pat1
+ and
snm1
+ genes. The
pac1
+ gene encodes a protein that is structurally similar to RNase III from
Escherichia coli
, but its normal function is unknown. We report here the purification and
characterization of the Pac1 protein after overexpression in
E.coli
. The purified protein is a highly active, double-strand-specific endoribonuclease that converts long double-stranded RNAs into short oligonucleotides and also cleaves a small hairpin RNA substrate. The Pac1 RNase
is inhibited by a variety of double- and single-stranded polynucleotides, but polycytidylic acid greatly enhances
activity and also promotes cleavage specificity. The Pac1 RNase produces 5
'
-phosphate termini and requires Mg
2+
; Mn
2+
supports activity but causes a loss of cleavage specificity. Optimal activity
was obtained at pH 8.5, at low ionic strength, in the presence of a reducing
agent. The enzyme is relatively insensitive to
N
-ethylmaleimide but is strongly inhibited by ethidium bromide and vanadyl
ribonucleoside complexes. The properties of the Pac1 RNase support the hypothesis that it is a eukaryotic
homolog of RNase III.
Double-strand-specific ribonuclease activities (dsRNases) have been described from
a variety of prokaryotic and eukaryotic sources, but few have been
characterized in detail (
1
). The archetype of this class of enzymes is RNase III from
Escherichia coli
(
2
-
4
). RNase III is an endonuclease that usually makes staggered cuts in both
strands of a double helical RNA, but in some cases it cleaves once in a single-stranded bulge in the helix (
5
,
6
).
In vitro
, the enzyme will degrade synthetic dsRNA to small oligonucleotides (
7
-
9
). Its primary biological function is the specific processing of rRNA and mRNA
precursors (
10
,
11
); but it has also been implicated in other diverse phenomenon, such as mRNA
turnover (
4
), conjugative DNA transfer (
12
), and antisense RNA-mediated regulation (
13
,
14
). The growing list of apparent homologs in the data bases (
15
,
16
) indicate that RNase III is highly conserved in both structure and function in
bacteria. There have been many reports of dsRNase activities in eukaryotic
cells, some of which exhibited properties consistent with a role in pre-rRNA processing (
17
,
18
), but the structure and biological significance of these enzymes is not known.
The best candidates for eukaryotic RNase III homologs are the Rnt1 RNase from
Saccharomyces cerevisiae
(
19
), which is an essential pre-rRNA processing enzyme, and the Pac1 RNase from
Schizosaccharomyces pombe
(
20
,
21
).
The
S.pombe
pac1
+
gene was first identified by virtue of its ability to induce sterility in wild-type cells (
20
) or in a
pat1
mutant (
21
), which spontaneously initiates sexual development at the restrictive
temperature (
22
,
23
). We isolated
pac1
+
as a multi-copy suppressor of
snm1
(
15
), a mutant that maintains reduced steady-state levels of several small nuclear RNAs (snRNAs) (
24
). This genetic evidence suggests a role for
pac1
+
in sexual development and snRNA metabolism, but its precise functions have not
been determined. The
pac1
+
gene encodes a protein of 363 amino acids whose carboxyl-terminal (C-terminal) two-thirds is very similar to
E.coli
RNase III (
20
,
21
). We showed that mutations that inactivate RNase III also abolish function when
reproduced in the
pac1
+
gene (
15
), which suggested a functional similarity between Pac1 and RNase III.
Consistent with these genetic findings, expression of
pac1
+
in
E.coli
produced an activity that converted dsRNA into acid soluble products (
21
); however, the double-strand specificity and other characteristics of this activity were not
determined. To better understand the enzymatic behavior of the Pac1 RNase, we
purified a poly-histidine-tagged version of Pac1 after overexpression in
E.coli
. We describe here the general biochemical properties of this enzyme, which is a
highly active, double-strand-specific endoribonuclease.
To create the tagged
pac1
+
gene we amplified the Pac1 coding sequence by PCR with the following primers
(NYU Pathology oligo service): 5'-ctt
We transformed BL21(DE3)pLysS [(F
-
ompT hsdS
B
(r
B
-
m
B
-
)
gal dcm
(DE3) pLysS(Cm
R
)] (Novagen) with pRSETpac and a single clone was grown at 30oC in 250 ml of tryptone-phosphate broth and induced with isopropylthio-[beta]-d-galactoside (IPTG) exactly as described by
Moore
et al
. (
26
). Three hours after induction, cells were collected by centrifugation and
stored at -70oC. A native protein extract was prepared from the frozen cells and
10 ml was fractionated on a 1.8 ml Pro-Bond Ni-affinity column. Both extract preparation and chromatography were
according to the instructions of the Xpress system kit (Invitrogen). The column
was washed until the A
280
was <0.01. Bound protein was eluted by consecutive application of 5 ml of elution
buffer containing 50, 200, 350, and 500 mM imidazole. Fractions (1 ml) were
collected and assayed for tagged Pac1 protein (tPac1) by Western blotting with
an anti-T7-tag monoclonal antibody (mAb) (Novagen). tPac1 eluted in two peaks
at 350 and 500 mM imidazole, which were pooled, concentrated with a Centricon-10 (Amicon) to a volume of 0.5 ml, diluted to 1.5 ml with storage buffer
to a final composition of 500 mM NaCl, 20 mM sodium phosphate (pH 7.4), 67 mM
imidazole, 1 mM dithiothreitol (DTT), 1 mM (ethylenedinitrilo)tetra-acetic acid (EDTA), and 30% glycerol, and stored at -20oC. The protein concentration measured with the Protein-Gold reagent (Integrated Separation Systems) and bovine serum albumin as the standard was 180 [mu]g/ml. The purified enzyme has remained active for ~1 year but loses activity upon further
dilution. A mock purification was performed on cells expressing the vector
alone. The 350 and 500 mM imidazole fractions from the Ni-affinity column were monitored by Western blotting with an anti-RNase III serum (gift of B. Simons, UCLA) and were assayed for RNase
III activity (
27
) with a
32
P-labeled dsRNA.
To prepare anti-Pac1 serum,
E.coli
transformed with pRSETpac (see above) were grown at 37oC in 500 ml of LB broth (GIBCO BRL) containing 100 [mu]g/ml ampicillin and 34 [mu]g/ml chloramphenicol to an OD
600
of 0.5, when tPac1 expression was induced with 0.6 mM IPTG. Three hours after
induction, cells were collected by centrifugation, washed with 15 ml of 50 mM
Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, and stored at -70oC. Frozen cells (3 g) were resuspended in 15 ml of lysis
buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1 M NaCl, 30 mM Tris-HCl, pH 8, 1 mM DTT, 1 mM EDTA); MgCl
2
(5 mM) and DNase I (10 [mu]g/ml) were added, and the suspension was incubated on ice for 10 min.
Inclusion bodies were collected by centrifugation at 10 000
g
for 15 min, washed four times with lysis buffer and twice with 50 mM Tris-HCl (pH 8), 5 mM MgCl
2
, 1 mM DTT, dissolved in 4.5 ml of loading buffer (
28
), and boiled for 10 min. One ml was fractionated by sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% gel. The tPac1 band,
visualized by staining with 0.05% Coomassie brilliant blue R250 in water, was
excised and sent to Cocalico Biologicals, Inc. (Reamstown, PA) for production
of antibodies in rabbits.
For Western blot assays, proteins were separated by SDS-PAGE and transferred to
nitrocellulose by semidry electroblotting (Owl Scientific). Blots were
incubated with anti-T7-tag mouse mAb (1:10
4
dilution), anti-Pac1 rabbit serum (1:5 * 10
3
), or anti-RNase III rabbit serum (1:10
4
). Immune complexes were detected with alkaline-phosphatase-conjugated goat anti-mouse IgG (H+L; 1:10
4
) or goat anti-rabbit IgG (Fc; 1:10
4
) (Promega) and the CSPD chemiluminescence substrate according to the
instructions of the Western-Light kit (Tropix). As controls, pre-immune rabbit serum replaced the anti-Pac1 and anti-RNase III sera or no first antibody was used.
The individual strands of the T3/T7 dsRNA (Fig.
1
B) were transcribed from a template produced by PCR amplification of the fourth
intron of the
S.pombe
[beta]-tubulin gene (
29
; gift of M. Yanagida, Kyoto, Japan) with the following primers (synthesized by
S. Teplin, Cold Spring Harbor Laboratory): 5'-gctcggaattaaccctcactaaag*ggaacGTAGGTTTTTTTGCTTTC-3', (T3 promoter in lower case, 5' end of the intron in upper case) and 5'-ggtacctaatacgactcactatag*ggagaCTACAGTCGTCAGTAC-3' (T7 promoter in
lower case, complement of the 3' end of the intron in upper case). The transcription initiation sites are
followed by asterisks. The PCR product was purified (
30
) and 40 ng was used in 20 [mu]l transcription reactions containing 40 mM Tris-HCl (pH 7.9), 6 mM MgCl
2
, 2 mM spermidine, 10 mM DTT, (50 mM NaCl for T3 reaction), 0.5 mM of each
ribonucleoside triphosphate, 50 [mu]Ci [[alpha]-
32
P]UTP (800 Ci/mmol; DuPont NEN), 20 U RNasin (Promega), and 20 U T3 or T7 RNA
polymerase (Ambion) at 37oC for 1 h. The transcripts were prepared by DNase I (Promega) digestion,
phenol extraction, and ethanol precipitation. The complementary RNAs were annealed and the unpaired ends removed by digestion
with RNases A and T1 (Ambion) (
31
). The hybrid was gel-purified (
32
) and stored in diethyl pyrocarbonate(DEPC)-treated distilled water at -20oC. The RNase III substrate was a 63 bp dsRNA synthesized from
pBluescript KS (Statagene). The N6 hairpin RNA (Fig.
1
B) was transcribed according to Milligan
et al
. (
33
) in a 20 [mu]l reaction containing 20 [mu]Ci [[alpha]-
32
P]UTP (800 Ci/mmol), 1.25 pmol of synthetic DNA template (gift of A. Nicholson,
Wayne State University, Detroit), and each ribonucleoside triphosphate at 1 mM
and purified as described above. The purified N6 RNA was renatured by
dissolving in 0.1 ml of 25 mM NaCl, 30 mM Tris-HCl (pH 7.6), heating at 90oC for 5 min, followed by slow cooling to room temperature and
storage at -20oC. The radioactive RNAs were quantified by liquid scintillation
counting in ScintiVerse BD (Fisher) and their concentrations calculated based
on the specific activity of the labeling nucleotide and the expected number of
radioactive phosphates in the RNA.
Standard conditions were 30 mM Tris-HCl (pH 7.6), 5 mM MgCl
2
, 1 mM DTT at 30oC. See figure legends for details of individual reactions. A theoretical
relative molecular mass (M
r
) of 45 504 was used to calculate tPac1 concentration. tPac1 was diluted in
reaction buffer immediately prior to the start of the reaction. Reactions were
assembled on ice and were started by the addition of 0.1 vol 50 mM MgCl
2
. Polycytidylic acid [poly(C), Pharmacia] was added as indicated in the figure
legends. Its concentration was calculated based on information supplied by the
manufacturer on the average polymer length. For trichloroacetic acid (TCA)
solubility assays, the reactions were stopped by addition of 0.5 ml of ice cold
5% TCA, and incubated on ice for 15 min. Aliquots (0.5 ml) were centrifuged at
16 000
g
for 4 min in a Spin-X filter unit (Costar). Aliquots of the filtrate (0.4 ml) were quantified
by liquid scintillation counting. For PAGE assays, reactions were stopped on
ice by addition of an equal volume of 80% formamide, 0.1% xylene cyanol, 0.1%
bromophenol blue, 12 mM EDTA. Aliquots (10 [mu]l) were fractionated by electrophoresis on a polyacrylamide/7 M urea gel and
the products detected by autoradiography. RNAs were excised and their
radioactivity quantified by liquid scintillation counting.
To determine the optimal pH, product formation was assayed in Mes (2-[
N
-morpholino]ethanesulfonic acid), Bes (
N
,
N
-bis[2-hydroxyethyl]-2-aminomethanesulfonic acid), Hepes (
N
-[2-hydroxyethyl]piperazine-
N
'-[2-ethanesulfonic acid]), Tris (tris[hydro- xymethyl]aminomethane), and Ches (2-[
N
-cyclohexyl]aminoethanesulfonic acid) at the following pH values: Mes, 5.5,
6.0, and 6.5; Bes, 6.5, 7.0, and 7.5; Hepes, 7.0, 7.5, and 8.0; Tris, 7.5, 8.0,
and 8.5; Ches, 8.5, 9.0, and 9.5.
To analyze the 5' ends of the Pac1 cleavage products, a 0.5 ml reaction containing 50 nM
N6 RNA labeled with [[alpha]-
32
P]GTP, 8 nM tPac1, 400 nM polycytidylic acid [poly(C)] (Pharmacia), 30 mM Tris-HCl (pH 7.5), 1 mM DTT, and 5 mM MgCl
2
was incubated for 30 min at 30oC. The RNA was collected by ethanol precipitation and fractionated by
denaturing PAGE in a 12% gel. The three major products were excised, eluted, and co-precipitated with 10 [mu]g of
E.coli
tRNA (Boehringer-Mannheim). One half of each product was digested with 5 U RNase T1, 0.25 [mu]g of RNase A, and 0.5 U RNase T2 (Calbiochem) in 5 [mu]l 10 mM Tris-HCl (pH 7.5), 1 mM EDTA at 37oC for 2 h. The digestion products were separated by
thin layer chromatography (TLC) on polyethyleneimine(PEI)-cellulose (Fisher) in
1.75 M ammonium formate (pH 3.5) (
34
) along with unlabeled nucleotide mono-, di-, and tetraphosphate markers (Sigma). The markers were visualized by
ultra-violet absorbance and the radioactive products by autoradiography with an intensifying screen. 5'-triphosphate-3'-monophosphate nucleotides were identified
by PEI-cellulose TLC in 0.75 M potassium phosphate (pH 3.5) (
35
). For secondary analysis of the presumed [
32
P]pGp digestion product, the spot was eluted from the TLC plate (
36
), mixed with 5 [mu]g of
E.coli
tRNA, and digested with 5 [mu]g nuclease P1 (Calbiochem) in 20 mM sodium acetate (pH 5.4) for 2 h at 37oC. P1 products were analyzed by PEI-cellulose TLC in 1 M LiCl (
37
).
The alignment of RNase III-like sequences shown in Figure
8
was assembled by manually adding new sequences to and extending our previously
published alignment (
15
). New sequences were gleaned from the literature or found by a BLAST (
38
) search performed at the NCBI using the BLAST network service. The BLAST
computation also revealed the repetition of blocks A and B in the unknown open
reading frames from
S.pombe
and
Caenorhabditis elegans.
To facilitate purification of the
S.pombe
Pac1 RNase, we inserted a tag between the first and second codons in the
pac1
+
gene. The tag encodes 35 amino acids that include six contiguous histidines, as
a nickel affinity ligand (His-tag), and part of the bacteriophage T7 gene 10 major capsid protein, as an
epitope tag (T7-tag) (Fig.
1
A). Expression of the tagged gene under the control of the normal
pac1
+
promoter in
snm1
(
24
) and
pat1
(
22
,
23
) mutant
S.pombe
strains fully complemented their temperature sensitive growth defects. Thus,
the tag does not impair Pac1 activity. To express the tagged protein (tPac1) in
E.coli
BL21(DE3)pLysS under the control of the inducible phage T7 promoter (
39
), we employed a method designed to produce soluble recombinant proteins (
26
). With this procedure ~50% of the tPac1 was soluble 3 h after induction with IPTG (Fig.
2
A, lanes 3 and 4), while at later times after induction the protein became
insoluble and extensive proteolytic breakdown occurred (not shown). Soluble
tPac1 was purified from cell lysates by Ni-affinity chromatography (
40
). The purified protein migrates as a single prominent band with a M
r
of ~50 000 by SDS-PAGE (Fig.
2
A, lane 5). The apparent M
r
, which is larger than the calculated molecular mass for tPac1 of 45.5 kDa, is
consistent with the electrophoretic behavior of an untagged Pac1 protein
expressed in
E.coli
(
21
) and identical to that obtained for tPac1 expressed in
S.pombe
(not shown). Purified tPac1 reacted with an anti-Pac1 serum (Materials and Methods) and with a mAb against the T7-tag (Fig.
2
B). The tPac1 preparation contains several smaller polypeptides as minor
components (Fig.
2
A, lane 5). All of these were recognized by the anti-Pac1 serum (Fig.
2
B, lane 2), while only the largest reacted with the anti-T7-tag mAb (Fig.
2
B, lane 1), which suggests the smaller polypeptides are proteolytic breakdown
products that have lost the epitope tag. If Pac1, like RNase III (
27
), is a dimer in its native state, polypeptides that lack the N-terminal tag might have bound to the column by association with the full
length protein.
We initially assayed tPac1 by a modification of the standard TCA solubility
assay used for RNase III (
41
). The substrate for these experiments was an internally labeled 101 bp (A+U)-rich dsRNA (T3/T7 dsRNA, Fig.
1
B). tPac1 was able to convert the T3/T7 dsRNA to acid soluble products under the
reaction conditions established for
E.coli
RNase III (
27
): 30 mM Tris-HCl (pH 7.6), 250 mM potassium glutamate, 10 mM MgCl
2
, 5 mM spermidine, 1 mM DTT, 0.1 mM EDTA, and 0.4 mg/ml
E.coli
tRNA. These conditions were, however, very inefficient-only 30% of the input RNA was rendered acid soluble even when tPac1 was ~80-fold in excess of the substrate. To determine whether this
activity was double-strand specific, we compared the degradation of the T3 strand, as a single-stranded substrate (ssRNA), against the T3/T7 dsRNA over a range of
monovalent cation concentrations. This experiment (data not shown) demonstrated
that at the lowest salt concentration the tPac1 RNase has a 10-fold preference for the T3/T7 dsRNA over the ssRNA substrate. Cleavage of
the dsRNA was inhibited with increasing salt concentration. This inhibition was
the same with the chloride and glutamate salts of potassium, which suggested
that the cation was the inhibitory component. A divalent cation was essential,
as no cleavage of either ds- or ssRNA was observed in the absence of MgCl
2
. These experiments indicated that the RNase III conditions were not ideal for
the tPac1 RNase. We therefore systematically altered the reaction components
and temperature to achieve the highest dsRNase activity. This preliminary
analysis yielded the following optimal reaction conditions: 30 mM Tris-HCl (pH 7.6), 1 mM DTT, 5 mM MgCl
2
at 30oC. Under these conditions dsRNase activity could be detected at tPac1
concentrations as low as 1 nM, and complete cleavage of a 20-fold molar excess of substrate was achieved in 10 min. We detected no
dsRNase activity under RNase III conditions at tPac1 concentrations <40 nM. This result is consistent with the conclusion that
E.coli
RNase III is not a significant contaminant in our tPac1 preparation.
We used a gel assay to visualize the cleavage pattern produced by the tPac1
RNase. Cleavage of the T3/T7 dsRNA at three different tPac1 concentrations is
shown in Figure
3
(lanes 2-5). At the lowest enzyme concentration (2 nM) nearly all of the T3/T7
dsRNA was converted to oligonucleotide products ranging in size from ~10 to 40 nt (lane 3). At higher enzyme concentrations the larger
oligonucleotides were converted to 10-20 nt products (lanes 4 and 5). In contrast, the T7 and T3 single-stranded RNAs were poor substrates for the tPac1 RNase (lanes 6-13). A quantitative analysis of the cleavage efficiency
revealed that 90% of the T3/T7 dsRNA was converted to oligonucleotide products
at the lowest enzyme concentration, while cleavage of the T7 and T3 ssRNAs was
detected only when tPac1 was 3-30-fold in excess of substrate (lanes 8, 9, 12 and 13). Different
cleavage efficiencies were obtained with the two ssRNAs, which may reflect
differences in secondary structure.
To re-examine the inhibition of tPac1 RNase activity by monovalent cations, we
assayed N6 RNA cleavage at several different NaCl concentrations. A 30 s
reaction time was used to measure the initial reaction velocity. As seen with
the T3/T7 dsRNA, N6 RNA cleavage was also inhibited by monovalent cations (Fig.
4
, lanes 8-13). Substrate loss and product formation steadily decreased with
increasing NaCl concentration. This experiment was repeated in triplicate and
quantified by measuring formation of the major product (the fragment migrating
slightly above the 34 nt DNA marker). Enzyme activity was barely detectable at
100 mM NaCl, while inhibition was complete at 200 mM. The NaCl concentration at
half maximal inhibition was 75 mM. We observed nearly identical inhibitory
effects with KCl and NH
4
Cl (not shown).
To test whether other divalent metal ions can substitute for the Mg
2+
requirement observed in our initial experiments, we assayed N6 RNA cleavage in
reactions in which Mg
2+
was replaced by Ca
2+
, Zn
2+
, Co
2+
, Mn
2+
, and Ni
2+
. For this experiment we added poly(C), which enhances the activity and cleavage
specificity of the tPac1 RNase with the N6 RNA substrate (see below). No
cleavage of N6 RNA was observed without a divalent cation (Fig.
5
, lanes 4 and 5). In the presence of MgCl
2
three major products are formed (lanes 6 and 7). [Compare this pattern with the
multiple cleavages in the absence of poly(C) shown in Figure
4
.] Only Mn
2+
could substitute for Mg
2+
, but tPac1 RNase activity was reduced, compared with the Mg
2+
reaction, and the specificity promoted by poly(C) was lost. The requirement for
a divalent cation was not relieved by the polycation spermidine (not shown).
To examine the pH optimum for tPac1 activity, we assayed the initial rate of N6
RNA cleavage at pH values between 5.5 and 9.5 in reactions buffered by Tris and
the zwitter ionic buffers Mes, Bes, Hepes, and Ches. Three pH values-below, above, and near the pK
a
-were assayed for each buffer. The tPac1 RNase exhibited a broad optimum
between pH 8 and 9. We obtained the highest enzyme activity at pH 8.5 in Ches
buffer; no activity was observed above pH 9.5 or below pH 6.5 (data not shown).
Some differences among the buffers were observed. For example, the enzyme
activity was slightly lower in Tris compared with the same pH in Ches or Hepes,
and Bes was inhibitory.
The Pac1 coding sequence predicts a single cysteine residue located at a
position that is part of the hydrophobic core of the double-stranded RNA binding domain (dsRBD) (
16
). To test if the sulfhydryl group in the unique cysteine is required, we
assayed the effect of
N
-ethylmaleimide (NEM) on tPac1 RNase activity. This experiment confirmed
that DTT is required for optimal tPac1 RNase activity and also showed that NEM
was only mildly inhibitory (~60% inhibition at 10 mM). We found that 100 [mu]M ethidium bromide caused complete inhibition of the tPac1 RNase,
consistent with the enzyme's preference for dsRNA. No inhibition was observed
at or below 0.1 [mu]M, and half maximal inhibition was at ~3 [mu]M. Vanadyl ribonucleoside complexes (VRC) are potent inhibitors of
ribonucleases that are presumed to mimic the scissile phosphodiester bond in
its trigonal bipyramid transition state (
42
). We found that VRC caused complete inhibition of tPac1 at 10 [mu]M and exhibited half maximal inhibition at ~2 [mu]M.
The strong preference of the tPac1 RNase for dsRNA over ssRNA substrates
prompted us to investigate the inhibitory properties of synthetic double- and single-stranded polynucleotides. Our initial experiments showed that the
double stranded homopolymer poly(I)-poly(C) was a potent inhibitor of the T3/T7 dsRNA cleavage by tPac1.
Consistent with the enzyme's dsRNA specificity, we found that poly(C) was not
inhibitory, even at concentrations that were 100-fold in excess of substrate. In marked contrast, however, poly(I)
inhibited tPac1 RNase activity as well as poly(I)-poly(C). To try to resolve these conflicting results, we examined the
effect of a variety of polynucleotides on the rate of cleavage of N6 RNA. The
results of this experiment confirmed the inhibition by poly(I)-poly(C) and poly(I) from another manufacturer and showed that
homoribopolynucleotides of A, G and U, as well as the heteroduplex poly(rA)-poly(dT), inhibited tPac1 RNase activity. Three polynucleotides-poly(C), the mixed polynucleotide poly(U-C), and the DNA poly(dI)-poly(dC)-did not inhibit but actually simulated tPac1
RNase activity.
To examine the stimulatory effect of poly(C) in greater detail, we assayed tPac1
RNase activity with the N6 RNA substrate in the presence of increasing amounts
of poly(C). The results of this experiment (Fig.
6
A) demonstrate the dramatic enhancement of activity promoted by poly(C). Not
only was there a quantitative effect but there was also a qualitative change in
the cleavage pattern. In the absence of poly(C) the tPac1 RNase makes multiple
cleavages in the N6 RNA (Fig.
6
A, lane 3; see also Fig.
4
). However, when poly(C) is present at 4 nM or above (Fig.
6
A, lanes 4, 5, and 6, and Fig.
5
, lanes 6 and 7), the cleavage pattern becomes more specific. Three predominant
products are formed, indicative of two relatively precise cleavages in the
helical stem. (The differences in the product band intensities in Figures
5
and
6
A reflect the radioactive nucleotides in the substrates.) To measure the effect
of poly(C) on enzyme activity we excised the upper hairpin product (see below
for product assignments) from the gel shown in Figure
6
A and quantified its radioactivity by liquid scintillation counting. The results
plotted in Figure
6
B (filled circles) demonstrate a steady increase in the rate of product
formation with increasing poly(C) concentration. Since the amount of a
particular product can reflect both enzyme activity and the choice of cleavage
sites, we also assayed the percent of substrate cleaved (open circles) and
found that poly(C) significantly enhances the cleavage rate even at low
concentrations that do not promote increased cleavage specificity. Thus,
poly(C) stimulates tPac1 RNase activity independently of its ability to promote
more precise cleavage.
Cleavage of phosphodiester bonds by
E.coli
RNase III produces 5' phosphoryl and 3' hydroxyl ends on the products at the cleavage site (
7
-
9
). To determine whether the same was true for the tPac1 RNase, we purified the
three major cleavage products of [[alpha]-
32
P]GTP-labeled N6 RNA (Fig.
5
) and digested them with a combination of RNases A, T1 and T2, to reduce them to
nucleoside-3'-monophosphates (Np). One of the N6 products should release
the 5' terminal tetraphosphate pppGp of the substrate RNA. The cleavage product
that migrated between the 26 and 15 nt DNA markers (Fig.
5
) released a labeled nucleotide upon RNase digestion that co-migrated by TLC with a product produced by digestion of the N6 substrate
RNA (not shown). The migration of these digestion products relative to a ppppA
marker indicated that they represented the 5'-terminal pppGp nucleotide of N6 RNA (
36
). Thus, the intermediate size product of N6 cleavage by the tPac1 RNase is the
5' fragment. Labeling of this product was weaker with [[alpha]-
32
P]UTP (Fig.
6
A) than with [[alpha]-
32
P]GTP (Fig.
5
), while the reverse was true for the smallest N6 product. The largest product
is equally intense with either label. From the sequence of N6 RNA, these data
are consistent with the largest product being the upper hairpin fragment and
the smallest being the 3' fragment.
If the tPac1 RNase uses the same cleavage mechanism as RNase III, then the upper
hairpin and 3' fragment should release nucleoside-3',5'-diphosphates (pNp) from their 5' ends after RNase digestion. The major
labeled product released from the 3' fragment had a mobility by PEI-cellulose TLC that was very similar to
the GDP marker (Fig.
7
, lane 1), which tentatively identified it as pGp. The two minor spots could not
be unambiguously identified from their mobilities, and their quantities were
insufficient for further analysis. The upper hairpin product did not liberate a
labeled pNp upon RNase digestion (not shown). The presumptive pGp spot produced
from the 3' fragment (Fig.
7
, lane 1) was eluted and digested with nuclease P1 to remove the 3' phosphate. The P1 digestion product co-migrated by PEI-cellulose TLC with GMP (Fig.
7
, lane 2). These results established that the 3' product of N6 RNA cleavage by tPac1 has a 5' phosphoryl group attached to a guanosine at its 5' end. Therefore, cleavage of phosphodiester bonds by the
tPac1 RNase leaves a phosphate at the 5' position. If tPac1 cleaved N6 RNA at the same site as RNase III, we
would have expected a labeled Up mononucleotide as a digestion product of the 3' fragment. We did not, however, detect a Up spot (Fig.
7
, lane 1, and data not shown). These results and the electrophoretic mobility of
the 3' fragment relative to the DNA markers, are consistent with cleavage on
the 5' side of the last G in the N6 RNA. We have not mapped the second cleavage
site, which we presume lies on the 5' side of the stem in the secondary structure model of N6 RNA (Fig.
1
B).
The enzymatic properties of the Pac1 RNase are very similar to RNase III. Both
enzymes are double-strand-specific RNases that convert synthetic dsRNAs into short, acid
soluble oligonucleotides (
7
-
9
) and leave 5'-phosphates on their cleavage products. The unit definition for
RNase III is the amount of enzyme that will solubilize 1 nmol of acid
precipitable polynucleotide phosphorus per hour (
3
). Using this definition, we estimate the specific activity of our tPac1
preparation to be 5 * 10
5
U/mg protein, which compares favorably with the 1.9 * 10
5
U/mg value for the most active RNase III preparation published (
27
). Since tPac1 appears to be fully functional in
S.pombe
, we are confident that the data we have obtained with the tagged protein
accurately reflect the characteristics of the authentic Pac1 enzyme. The Pac1
RNase, like a dsRNase from calf thymus (
18
), differs from
E.coli
RNase III in its sensitivity to monovalent cations. In contrast, RNase III is
actually stimulated by moderate concentrations of monovalent cations (
2
). At low monovalent cation concentrations, RNase III loses cleavage specificity
(
3
,
6
,
27
). We have not observed similar effects for Pac1. Like most of the dsRNases
described (
1
), Pac1 has an absolute requirement for a divalent cation. Substitution of Mn
2+
for Mg
2+
causes Pac1 to lose cleavage specificity. A similar effect was reported for Mn
2+
and Co
2+
with
E.coli
RNase III (
27
). Despite the similarities in biochemical properties, Pac1 must possess
important structural differences compared with its bacterial counterpart since
our anti-Pac1 sera do not react with purified
E.coli
RNase III (G.R., unpublished observations) and anti-RNase III sera do not recognize tPac1 or the unmodified protein (
21
). With respect to functional equivalence, the
pac1
+
gene can not suppress the
rnc105
mutation in the
E.coli
RNase III gene (
rnc
) (
21
) or a null allele (G.R., unpublished results). The
rnc
gene has not been tested for its ability to cure the
S.pombe
snm1
and
pat1
mutants, which are complemented by
pac1
+
. However, overexpression of
rnc
in
S.cerevisiae
is lethal (
43
).
Under our standard conditions, the Pac1 RNase produces a cleavage pattern with
the N6 hairpin RNA that suggests a mixture of random cuts and specific
cleavages at preferred sites. However, when poly(C) is added 8-10-fold in excess of substrate, the cleavage pattern becomes much more
specific. This enhanced specificity is accompanied by an ~8-fold increase in enzyme activity as assayed by the rate of specific
product formation. The basis for these stimulatory effects is unclear. Two
other polynucleotides-the mixed ribopolymer poly(U-C) and the DNA poly(dI)-poly(dC)-produced similar stimulatory effects, while all other
polynucleotides tested were inhibitory. Perhaps Pac1 has an inherent affinity
for cytidine-containing polynucleotides. The inhibition by polynucleotides was not
consistent with Pac1's stringent preference for dsRNA substrates. For example,
some single-stranded polymers, such as poly(I), were as potent inhibitors of the Pac1
RNase as poly(I)-poly(C).
The biochemical properties of the Pac1 RNase imply a functional homology with
RNase III that is supported by the structural similarity. Pac1 is 25% identical
over its C-terminal two-thirds to
E.coli
RNase III (
20
,
21
), and this relationship extends to other bacterial and eukaryotic RNase III-like proteins (
15
). Figure
8
shows an alignment of 13 protein sequences that share a marked similarity with
RNase III. The sequences are arranged in three conserved blocks labeled A, B
and C. In each block the first nine sequences are bacterial and the last four
are eukaryotic. Blocks A and B surround the sites of the
rnc105
and
rnc70
mutations that inactivate both
E.coli
RNase III (
4
,
44
) and Pac1 (
15
). Block A is the N-terminal end of the RNase III similarity. The actual N-termini of the bacterial proteins lie ~20 amino acids upstream of the left end of block A. The N-termini of Pac1 and its
S.cerevisiae
homolog, Rnt1p (
19
), lie ~100 amino acids upstream. The last two sequences in Figure
8
(
Sp
orf and
Ce
orf) are part of very large open reading frames of unknown function in
S.pombe
and
C.elegans
. Their N-termini lie >1000 amino acids upstream. The C-termini of all the sequences lie not far beyond the right end of
block C.
The glycine at the
rnc105
position in block A is conserved in all the sequences, and the adjacent amino
acids form the most highly conserved stretch in the RNase III-like proteins. Block B surrounds the
rnc70
site, which is glutamic acid in all the sequences except that from
Bacillus subtilis
(
Bs rnc
), where it is replaced by lysine. A lysine at this position is surprising since
the
rnc70
mutation converts the conserved glutamic acid to a lysine. Given that a
transition at the first position is sufficient to convert a glutamic acid codon
to one for lysine, a re-examination of the
B.subtilis
sequence may be warranted. Block C represents the putative dsRBD, which is
required for the function of
E.coli
RNase III (
44
) and Pac1 (
15
). The bacterial dsRBD sequences, with the exception of that from
Mycoplasma genitalium
(
Mg rnc
), are clearly related and are good matches to the dsRBD consensus. Since the
other
Mycoplasma
sequence shown [
M
(sp) orf] and another not shown (
16
) align well with the other bacterial sequences, the deviation in the
M.genitalium
sequence may be the result of a sequencing error that generated a frame shift.
As noted previously (
45
), the Pac1 dsRBD is not a good match to the consensus except for the 20 amino
acids at the C-terminus. This appears to hold true for the other eukaryotic sequences.
The unknown
S.pombe
orf, however, deviates in an important way near its C-terminus. A pair of alanines in this region are found in nearly all dsRBDs
(
46
), and changing these alanines to valines abolishes Pac1 function (
15
). This result is consistent with the structural models of the dsRBD, which
predict that bulky side groups are not tolerated at these positions (
16
,
47
). Thus, the absence of this alanine pair in the
S.pombe
orf suggests either a sequencing error or a protein with poor dsRNA binding properties.
The two orfs of unknown function from
S.pombe
and
C.elegans
are more closely related to one another than they are to other RNase III
sequences. These orfs predict proteins with interesting dual functions. The
RNase III-like domain is contained within ~300 amino acids at the C-terminal end of each protein. Blocks A and B are separated by a
variable number of amino acids not found in the other RNase III sequences. In
addition, blocks A and B, but not C, are repeated immediately upstream of the
complete RNase III-like domains. This reiteration is indicated by the upper of the two lines
of sequence shown for the
S.pombe
and
C.elegans
orfs in blocks A and B in Figure
8
. The large (900-1200 amino acids) N-terminal regions of the two orfs share a similarity (not shown)
with the DEAD box family of RNA-dependent ATPases/helicases (
48
). The canonical DEAD motif is replaced by the sequence DECH in the
S.pombe
and
C.elegans
orfs. A similar duality of structure is seen in the human RNA helicase A and
Drosophila
maleless
proteins, each of which has two N-terminal dsRBDs upstream of a central DEAH helicase domain (
49
). The unusual structure of the
S.pombe
and
C.elegans
orfs suggests enzymes that can both unwind and cleave dsRNA. Eukaryotic cells
may, therefore, have more than one type of RNase-like dsRNase.
S.cerevisiae
Rnt1p is a pre-rRNA processing enzyme (
19
). The similarity between Rnt1p and Pac1 suggests that the
S.pombe
enzyme might also participate in rRNA maturation. Since a
pac1
null mutation is lethal (
21
), the second RNase III-like gene in
S.pombe
may not be able to substitute for Pac1's essential function in rRNA synthesis.
Conversely, the observed effects of Pac1 overexpression on fertility (
20
,
21
) and snRNA metabolism (
15
) may reflect its ability to compensate for loss or normal attenuation of the
function of the large
S.pombe
RNase III-like protein.
The retention of an RNase III gene in the minimal genome of
Mycoplasma genitalium
(
50
) and its conservation in bacteria and eukaryotes implies an ancient and
important function. The possibility of multiple forms of RNase III and the
emerging involvement of dsRNA in cell growth, differentiation, and development
(
1
,
51
) raise the possibility that RNase III-like enzymes play critical regulatory roles in eukaryotic cells.
We thank Allen Nicholson, Bob Simons, Ron Beavis, Mitsuhiro Yanagida and David
Beach for generous gifts of materials and strains, Naoko Tanese for crucial
advice on protein expression in
E.coli
, Warren Jelinek for assistance in the preparation of TEAB and, along with Jim
Borowiec, for critical comments on the manuscript, and Pam Cowin for cogent
editorial suggestions. We thank Manuel Ares for communication of results before
publication. This work was supported by Bridging Funds from the New York
University Medical Center and by Developmental Funds from the Center for AIDS
Research at NYU. D.F. was supported by a Whitehead Fellowship for Junior
Faculty and an Irma T. Hirschl Career Scientist Award.
REFERENCES
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