From the Institute of Biological Sciences and
§ Gene Experiment Center, University of Tsukuba,
Tsukuba-shi, Ibaraki 305, Japan and the
Department of
Biochemistry, Mount Sinai School of Medicine of the City University
of New York, New York 10029-6574
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ABSTRACT |
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Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5'- and 3'-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNase III. The B. subtilis enzyme that has similar activity to Escherichia coli RNase III has been purified and is designated Bs-RNase III. Recently, B. subtilis rncS has been shown to encode Bs-RNase III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379-7385). We show here that Bs-RNase III and the purified His-tagged product of rncS cleave pre-scRNA at both 5'- and 3'-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3'-site is less efficient. The 5'-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3'-end. Bs-RNase III cleavage yields a two-base 3'-overhang, which is consistent with the manner in which E. coli RNase III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNase III is an enzyme that processes pre-scRNA.
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INTRODUCTION |
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The mammalian signal recognition particle
(SRP),1 which targets the
presecretory protein to the endoplasmic reticulum membrane, is composed
of a 7SL RNA (here referred to as SRP RNA) and six proteins (1, 2).
Eukaryotic SRP RNAs consist of four functional domains (domains I-IV).
SRP RNA homologues have been identified in eubacteria, including
Escherichia coli and Bacillus subtilis (4.5S RNA
for E. coli and small cytoplasmic RNA (scRNA) for B. subtilis) (3, 4). In vivo, eubacterial SRP RNA binds to Ffh protein, which is homologous to eukaryotic SRP54 protein, an SRP
subunit that binds to signal sequences (3, 5). Like mammalian SRP, the
E. coli 4.5S RNA-Ffh complex binds specifically to signal
sequences (6), whereas B. subtilis Ffh can bind signal sequences by itself (7). Moreover, proteins homologous to the -subunit of mammalian SRP receptor (SR
) have been identified in
E. coli (FtsY) and B. subtilis (Srb) (8-10).
In vitro and in vivo analyses demonstrate that
bacterial SRP RNA-Ffh-FtsY (Srb) mimics an SRP pathway in eukaryotes
(11-13). On the other hand, E. coli 4.5S RNA and B. subtilis scRNA are involved in mRNA translation on ribosomes.
In addition, 4.5S RNA and scRNA also bind to protein synthesis
elongation factor G, and they may be involved in elongation factor G
recycling (14).
The predicted secondary structure of B. subtilis scRNA is
strikingly similar to that of eukaryotic SRP RNA, although it lacks domain III (3, 4). It is transcribed as a 354-nucleotide (nt) primary
transcript and then processed to a 271-nt mature scRNA (15). This
maturation requires processing at both 5'- and 3'-sites. Moreover, 5'-
and 3'-flanking sequences of scRNA precursor (pre-scRNA) constitute a
stable stem structure based on complementary sequences encompassing the
processing sites. Therefore, both processing sites should be located
within a double-stranded stem structure. Ribonuclease III (RNase III)
of E. coli is a double-stranded RNA (dsRNA)-specific
ribonuclease, and it processes several RNAs, including rRNA precursor,
T7 early gene transcripts, and PL transcript
(16-20). RNaseIII processing can affect mRNA half-life and the
level of gene expression (21), suggesting that processing by RNase III
is biologically important. In eukaryotic cells, ribonucleases with
RNase III-like activity are Rnt1p of Saccharomyces
cerevisiae and PacIp of Schizosaccharomyces pombe (22,
23). Panganiban and Whitely (24) discovered an enzymatic activity in
B. subtilis that is similar to E. coli RNase III,
and they called it Bs-RNase III. Purified Bs-RNase III cleaves B. subtilis rRNA precursor and early transcripts of B. subtilis bacteriophage SP82 (24, 25).
We found that the srb gene is the last gene in a three-gene operon, the first gene of which encodes rncS, the product of which shares 36% amino acid identity with E. coli RNaseIII. Oguro et al. (13) cloned the rncS gene, and Wang and Bechhofer demonstrated that this gene codes for Bs-RNaseIII (26). The presence in an operon of a gene (rncS) coding for a double-stranded RNA processing enzyme and a gene coding for a component of the B. subtilis SRP receptor (srb), suggesting that Bs-RNaseIII might be involved in scRNA processing.
In this study, we examined the function of Bs-RNase III in pre-scRNA processing. The results demonstrated that Bs-RNase III is an essential endonucleotic agent that processes at both 5'- and 3'-sites. Additionally, purified His-tagged RncS protein can process pre-scRNA, and the cleavage sites are identical to those of Bs-RNase III. Moreover, unprocessed pre-scRNA accumulated in a B. subtilis strain containing a truncated rncS gene.
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MATERIALS AND METHODS |
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Plasmid Construction-- We constructed the E. coli plasmid pTUE962 expressing B. subtilis RncS tagged with hexahistidine at its C terminus (RncS-His6) as follows. The B. subtilis rncS gene was amplified by PCR using the primers 5'-GGTTACCATGGCAAAACACTCAC-3' and 5'-ACATAGATCTAATAAGACGGCATAC-3'. These primers were designed to generate NcoI and BglII (underlined) sites at the 5'- and 3'-ends, respectively, of the PCR product. The PCR product was digested with NcoI and BglII and then ligated into the NcoI and BglII sites of pQE60 (Qiagen Inc., Chatsworth, CA).
To prepare the scRNA precursor in vitro, a 396-nt fragment encoding the scRNA precursor was amplified by PCR using 5'-TATACTTAAGCTTGCATCG-3' and 5'-GTTTCGGATCCAAAAGCTC-3' (Fig. 1). These primers were designed to generate HindIII and BamHI sites (underlined) at the 5'- and 3'-ends, respectively, of the PCR product, which was purified, digested with HindIII and BamHI, and then ligated into the HindIII and BamHI sites of pSP64 (Promega, Madison, WI). The constructed plasmid was designated pTUE961. The construction of plasmid pJFD4, which was used for in vitro transcription of SP82 mRNA, has been described by Mitra and Bechhofer (27).In Vitro Synthesis of 32P-Labeled SP82 mRNA A
Site and Pre-scRNA--
Plasmids pJFD4 and pTUE961 were linearized by
digestion with XbaI and BamHI, respectively. The
transcription reaction mixture consisting of 40 mM Tris-HCl
(pH 7.9); 6 mM MgCl2; 10 mM NaCl; 0.5 mM each ATP, GTP, UTP; 2.5 µM CTP; 70 units of RNase inhibitor (Takara Shuzo Co., Ltd., Kyoto, Japan); 10 µCi of [-32P]CTP (400 Ci/mmol, Amersham Pharmacia
Biotech, Little Chalfont, UK); 1 pmol of the linearized DNA fragment;
and 35 units of SP6 RNA polymerase (Takara Shuzo Co., Ltd.), was
incubated at 37 °C for 1 h. RNAs were precipitated by ethanol
and then dissolved in sterile and deionized water.
Purification of Bs-RNase III and RncS Tagged with Hexahistidine-- Bs-RNase III was purified as described previously (27). The S30 cell extract, which was prepared from protease-deficient strain GP208, was precipitated by ammonium sulfate (65% saturation). The pellet was dissolved and was fractionated by phenyl-Sepharose and poly(I)·poly(C) agarose steps. Bs-RNase III fraction was purified based upon its processing activity toward B. subtilis SP82 phage mRNA A site.
RncS-His6 was expressed in E. coli M15 harboring pREP4 (Qiagen Inc.) incubated with shaking for 5 h at 37 °C in 150 ml of Luria-Bertani (LB) medium supplemented with 1 mM isopropyl-1-thio-RNase III Assay-- Cleavage of pre-scRNA was as described by Mitra and Bechhofer (27). The assay mixture (60 µl) containing 10 mM Tris-HCl (pH 7.9), 3 mM MgCl2, 100 mM NaCl, 0.04 pmol of labeled RNA, and 0.04 units of purified enzyme was incubated at 37 °C. The reaction was terminated by adding phenol/CH3Cl, and then the aqueous phase was precipitated with 2 volumes of ethanol. The recovered samples were separated by electrophoresis on an 8 M urea-6% polyacrylamide gel, which was then dried, exposed to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) and visualized by autoradiography.
Constructions of B. subtilis BG217 and BG218--
The
construction of strains BG217 and BG218 is described by Wang and
Bechhofer (26). To construct a disrupted rncS gene, a
600-base pair HindIII fragment located within the
rncS coding sequence was cloned into the HindIII
site of E. coli plasmid pDH88 (28) (plasmid pBSR18). The
control was a full-length rncS coding sequence amplified by
PCR that contained XbaI and BglII sites at its
5'- and 3'-ends, respectively. This sequence was cloned into the
XbaI-BglII sites of pDH88 (plasmid pBSR17).
Plasmid pBSR18 and pBSR17 were used to transform B. subtilis
BD170 (trpC2 thr-5) to yield the chloramphenicol-resistant
strain BG218 (rncS1) and BG217
(rncS+), respectively. As a result, BG218
expresses C-terminal truncated RncS (1-237 amino acids) lacking 29 amino acids and having an extra 9 amino acids derived from vector.
Primer Extension Analysis--
Total RNAs of B. subtilis BG217 and BG218 were extracted as described by Ambulos
et al. (29). Primers psc-1 (5'-AACCTCGAGAAGGGATTCG-3') and
psc-2 (5'-AACTCACCTAATATTAGG-3') were used to map the 5'- and 3'-ends,
respectively, of the cleavage sites in pre-scRNA (Fig. 1). Pre-scRNA
was synthesized in vitro as described above except
transcription proceeded in the presence of 0.5 mM of
nonradiolabeled CTP instead of [-32P]CTP. The primers
psc-1 and psc-2 were labeled at the 5'-end with T4 polynucleotide
kinase (Toyobo, Inc., Osaka, Japan) and [
-32P]ATP
(6000 Ci/mmol, Amersham International). Pre-scRNA (40 µg) cleaved
in vitro, or total B. subtilis RNA (100 µg) was
hybridized with the 32P-labeled oligonucleotide primers
(1 × 105 cpm) at 35 °C overnight. After
hybridization, samples were precipitated with ethanol and dissolved in
20 µl of reverse transcriptase buffer (50 mM Tris-HCl (pH
7.6), 60 mM KCl, 10 mM MgCl2, 1 mM each dNTP, 1 mM DTT). 40 units of
Rous-associated virus 2 reverse transcriptase (Takara Shuzo Co., Ltd.)
was then added, and the mixture was incubated at 42 °C for 1 h.
The samples were resolved by 8 M urea-6% polyacrylamide gel electrophoresis and visualized by autoradiography. The psc-1 primer
was also used for DNA sequencing.
Northern Hybridization-- Total RNAs of B. subtilis BG217 and BG218 were resolved by 8 M urea-6% polyacrylamide gel electrophoresis and blotted onto a GeneScreen Plus membrane (NEN Life Science Products). The membrane was then hybridized with 5'-end-labeled psc-1.
RNase Protection Assay--
A 192-base pair
EcoRI-BamHI fragment of pTUBE961, including the
3'-portion of the B. subtilis scRNA, was isolated and
inserted into pBluescript II SK (Stratagene, La Jolla,
CA) between the EcoRI-BamHI sites. T3 RNA
polymerase and [
-32P]CTP were used to synthesize a
radioactive probe encompassing the 3'-portion of the scRNA gene and to
which it is complementary. Total RNAs of BG217 and BG218 (10 µg each)
and 2 × 105 cpm of the 32P-labeled probe
were hybridized at 45 °C overnight, and then the mixture was
digested with RNase A (1 unit) and RNase T1 (200 units) at
37 °C for 30 min. The samples were resolved by 8 M
urea-6% polyacrylamide gel electrophoresis and visualized by
autoradiography.
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RESULTS |
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In Vitro Transcripts of the Cloned scRNA Gene--
Plasmid
pTUE961 contained a 382-nt fragment of PCR-amplified scRNA gene (Fig.
1). The BamHI-digest from
pTUE961 was used as a template for in vitro transcription by
SP6 RNA polymerase. The putative secondary structure of the in
vitro transcript is shown in Fig. 2.
The arrows in Fig. 2 indicate sites 5' and 3' processing site by Bs-RNase III. These processing sites exist beside the tetrad
loops and yield a two-base 5'-overhang. To prepare pre-scRNA as the
substrate(s) for Bs-RNase III, plasmid pTUE961 was linearized with
BamHI, and transcripts were obtained by a reaction of SP6 RNA polymerase in the presence of -32P-labeled CTP. As a
result, two major transcripts, S1 and S2, were obtained (Fig.
3 lanes 1 and 7).
Compared with a DNA molecular marker, the lengths of S1 and S2 were
estimated to be 389 and 366 nt, respectively. Because the scRNA gene
fragment in pTUE961 contains an endogenous
-independent
transcriptional terminator of scRNA (Figs. 1 and 2), S1 and S2
represent products terminated at the BamHI site and at the
-independent terminator, respectively.
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In Vitro Processing of scRNA Precursor by Bs-RNase III and RncS-His6-- To examine whether Bs-RNase III processes pre-scRNA, 32P-labeled pre-scRNA was treated in vitro with the purified Bs-RNase III. At 0.5 min of incubation with the purified Bs-RNase III, two processed bands (Fig. 3, lane 2, P1 and P2) appeared. After a prolonged incubation, S1 and S2 were converted into P1, P2, and P3 (Fig. 3, lanes 3-6). At 45 min of incubation, 33.3% of transcripts were converted into P3 product (Fig. 3, lane 6). Compared with a DNA molecular marker, the lengths of these products were estimated to be 340, 317, and 275 nt, respectively. To exclude the possibility of the contamination of other ribonucleases in purified Bs-RNase III, we also carried out the in vitro processing assay of pre-scRNA by purified RncS-His6. S1 and S2 were processed to P1 and P2, respectively, and finally to P3 in the same manner as with Bs-RNase III (Fig. 3, lane 8).
Determination of the Cleavage Sites of the scRNA Precursor-- We determined the constitution of the 5'- and 3'-ends of the processed products by primer extension using primers psc-1 and psc-2, respectively (Figs. 1 and 4). Nonlabeled pre-scRNA was transcribed from BamHI-digested pTUE961 in vitro. Pre-scRNA was primer-extended after an incubation with Bs-RNase III for 30 min at 37 °C. When psc-1 was used as the primer, a major band corresponding to the U residue at position 38 was detected (Fig. 4A, lane 2). This indicates that Bs-RNase III cleaved the 5'-site of pre-scRNA at the U---U bond of positions 37-38 (Fig. 1). The 5'-end of the processed pre-scRNA was identical to that of the mature scRNA obtained in vivo (15). The 3'-end of the processed product was also determined by primer extension using the primer psc-2, which anneals to a region in the 3'-flanking fragment at the positions 335-352 (Fig. 1). One distinct band appeared that was 40 nt long (Fig. 4B, lane 2). This indicates that the 5'-end of the 3'-flanking fragment from pre-scRNA corresponds to an A residue at position 313 and that the cleavage site at the 3'-site of P3 created by Bs-RNase III is located at the C-A bond of nucleotide 312-313. This 3'-end is 4 nucleotides downstream of the 3'-end of the mature scRNA obtained in vivo (15) (Fig. 1). Although both P1 and P2, which contain the 3'-flanking fragment, remained after a 30-min incubation (Fig. 3, lane 5), the extension products corresponding to P1 and P2 were not revealed by this assay (Fig. 4B). We suppose that they are too long (>300 nt) for detection by this gel system. Based on their length and the results of the primer extension, P1 and P2 are intermediate products that are processed at only the 5'-sites of S1 and S2, respectively. P3 is processed at both the 5'- and 3'-sites of S1 and S2. However, P3 contains four excess nucleotides at the 3'-end of mature scRNA. This 275-nt intermediate product is designated scRNA-275. We also examined primer extension with pre-scRNA which had been incubated with RncS-His6 for 30 min at 37 °C. When psc-1 and psc-2 were used as primers, we detected extension bands with the same molecular weight as those of the products of the pre-scRNA cleaved by Bs-RNase III (data not shown).
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Effect of Defective Bs-RNase III in Vivo--
To confirm that RncS
protein is involved in pre-scRNA processing in vivo, we
constructed the mutant strain BG218 (rncS1), which
expresses defective RncS lacking part of the C-terminal dsRNA binding
domain (26). We Northern blotted total RNAs from the control strain
BG217 and the rncS mutant strain BG218. Fig. 5 shows that significant amounts of
pre-scRNA accumulated in strain BG218. We then defined the 5'- and
3'-ends of scRNA in the BG217 and BG218 strains. To examine the 5'-end,
we performed primer extension with 5'-labeled psc-1 against total RNAs
of BG217 and BG218. For BG217, one distinct band was detected at the U
residue at position 38, which is the 5'-end of mature scRNA (Fig.
6A, lane 1). For BG218, the
major band at position 38 was present, but an additional band was
detected at the G residue at position 1, which is the 5'-end of
pre-scRNA (Fig. 6A, lane 2). We performed an RNase
protection assay to define the 3'-end. One major protected band of
132-nt was detected in BG217. This band corresponded with the A residue
at position 308, which is the 3'-end of mature scRNA (Fig. 6B,
lane 1). In contrast, two major protected bands of 132 and 178 nt
were detected in BG218. The additional 178-nt band corresponded with
the U residue at position 354, which is the 3'-end of pre-scRNA (Fig.
6B, lane 2). Furthermore, one minor band of 136 nt was
detected in BG218. This band corresponded with the C residue at
position 312, which is the 3'-end of scRNA-275 (Fig. 6B, lane
2). These results show that Bs-RNase III processes pre-scRNA
in vivo as it does in vitro.
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DISCUSSION |
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RNase III is an endonuclease that specifically cleaves dsRNA structures. In this study, we showed that Bs-RNase III processes pre-scRNA at both 5'- and 3'-sites. Pre-scRNA was processed to a 275-nt fragment (scRNA-275) upon incubation with Bs-RNase III or RncS-His6 in vitro. The 5'-end of the processed pre-scRNA was identical to that of mature scRNA. However, the 3'-end of processed pre-scRNA was 4 nucleotides downstream from that of mature scRNA (Fig. 1). These results suggest that pre-scRNA is processed to scRNA-275 by RNase III first, and then the four nucleotides are removed from the 3'-end of scRNA-275 by another RNase(s). The 3'-end of scRNA-275 was also detected by an RNase protection assay of the RNA preparation from the BG218 mutant strain as a minor band (Fig. 6B, small arrowhead). The staggered cleavage site of pre-scRNA by Bs-RNase III is at the internal tetrad loop (Fig. 2). Single cleavages by Bs-RNaseIII on SP82 sites A and B were also found to occur in an internal bulge sequence (27).
Struck et al. (15) indicated that 5'-end processing of pre-scRNA occurred prior to 3'-end processing in vivo. Fig. 3 shows that the processed products P1 and P2 appeared first, followed by P3 as the incubation period increased. At 10 min of incubation (Fig. 3, lane 5), almost all transcribed products (S1 and S2) were converted into P1, P2, and P3. However, the 5'-end-processed products, P1 and P2, did not disappear as the incubation period extended, and the amount of P3 did not significantly increase. The cleavage efficiency of the 3'-site is one-tenth that of 5'-site. This suggests that cleavage at the 3'-site determines the maturation efficiency of pre-scRNA or that another factor is necessary for efficient cleavage. We suppose that in vitro processing proceeds in the same manner as in vivo processing.
Recently, B. subtilis genomic DNA was completely sequenced (30). The BLAST computation showed that only the rncS product had significant homology with E. coli RNase III. Furthermore, we searched the B. subtilis orf data base for the amino acid sequence NERLEFLGD, which is conserved in RNase III of E. coli, S. cerevisiae, S. pombe, and B. subtilis. The BLAST computation showed that the yitV product had a similar sequence (PERLQFIGD; underlined residues show conserved amino acids). However, the full YitV amino acid sequence did not show significant homology with RNase IIIs. These data suggest that rncS is the only gene that codes for RNase III activity in B. subtilis. In B. subtilis strain BG218, which expresses a C-terminal truncated RncS, significant amounts of pre-scRNA accumulated in this strain (Fig. 5). The truncated RncS is missing a part of the dsRNA binding domain. We think that the C-terminally truncated RncS retains catalytic activity but has a decreased ability to bind dsRNA. As a result, pre-scRNA processing efficiency is decreased in BG218. We attempted to construct a null mutant of rncS, using integration vectors in which four different regions of the rncS gene were cloned, but these efforts were unsuccessful (data not shown). This suggests that Bs-RNase III is essential for viability of B. subtilis and that processing of RNA by Bs-RNase III plays an important role in B. subtilis.
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ACKNOWLEDGEMENT |
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We thank N. Foster for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants-in-aids for scientific research from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D64116.
¶ To whom correspondence should be addressed. Tel. and Fax: 81-298-53-6680; E-mail: kyamane{at}sakura.cc.tsukuba.ac.jp.
1 The abbreviations used are: SRP, signal recognition particle; scRNA, small cytoplasmic RNA; pre-scRNA, scRNA precursor; nt, nucleotide(s); scRNA-275, 275-nt intermediate form of scRNA; dsRNA, double-stranded RNA; Bs-RNase III, RNase from B. subtilis; rncS, B. subtilis gene encoding Bs-RNase III; PCR, polymerase chain reaction.
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REFERENCES |
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