JOURNAL OF BACTERIOLOGY, July 2010, p. 3279–3286
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 13
Initiation of Decay of Bacillus subtilis rpsO mRNA by
Endoribonuclease RNase Y?
Shiyi Yao and David H. Bechhofer*
Department of Pharmacology and Systems Therapeutics, Box 1603, Mount Sinai School of Medicine of
New York University, New York, New York 10029
Received 3 March 2010/Accepted 18 April 2010
rpsO mRNA, a small monocistronic mRNA that encodes ribosomal protein S15, was used to study aspects of
mRNA decay initiation in Bacillus subtilis. Decay of rpsO mRNA in a panel of 3?-to-5? exoribonuclease mutants
was analyzed using a 5?-proximal oligonucleotide probe and a series of oligonucleotide probes that were
complementary to overlapping sequences starting at the 3? end. The results provided strong evidence that
endonuclease cleavage in the body of the message, rather than degradation from the native 3? end, is the
rate-determining step for mRNA decay. Subsequent to endonuclease cleavage, the upstream products were
degraded by polynucleotide phosphorylase (PNPase), and the downstream products were degraded by the 5?
exonuclease activity of RNase J1. The rpsO mRNA half-life was unchanged in a strain that had decreased
RNase J1 activity and no RNase J2 activity, but it was 2.3-fold higher in a strain with decreased activity of
RNase Y, a recently discovered RNase of B. subtilis encoded by the ymdA gene. Accumulation of full-length rpsO
mRNA and its decay intermediates was analyzed using a construct in which the rpsO transcription unit was
under control of a bacitracin-inducible promoter. The results were consistent with RNase Y-mediated initiation
of decay. This is the first report of a specific mRNA whose stability is determined by RNase Y.
The rate of decay of an mRNA is important for determining
the level of gene expression. Studies on the mechanism of
mRNA decay in Escherichia coli have progressed based on
detailed knowledge of the RNases involved in the process and
construction of RNase mutant strains. In a generally accepted
model that applies to the turnover of many E. coli mRNAs, the
5?-end-dependent RNase E is responsible for the rate-deter-
mining endonuclease cleavage, which produces an upstream
fragment that is subject to 3?-to-5? exonucleolytic decay by
RNase II and a downstream fragment that is subject to further
RNase E endonucleolytic cleavage (5, 25). Recent studies have
suggested that, in some cases, a preliminary step in RNase E
binding is conversion of the native triphosphate 5? end, which
is a poor substrate for RNase E binding (23, 26, 40), to a
monophosphate 5? end by pyrophosphatase activity (4, 11).
Degradation from the 3? end can also occur, and this is depen-
dent on the 3? extending activity of poly(A) polymerase (17).
A similar level of understanding of the mechanism of
mRNA decay has not been achieved for the model Gram-
positive organism Bacillus subtilis. Sequence homologues of
some of the E. coli enzymes that play major roles in mRNA
decay [e.g., RNase E, RNase II, and poly(A) polymerase] can-
not be identified in the B. subtilis genome. Nevertheless, stud-
ies on a number of mRNAs, some of which are constitutively or
inducibly stable, have suggested that mRNA decay in B. subtilis
also initiates from the 5? end (9). B. subtilis polynucleotide
phosphorylase (PNPase), encoded by the pnpA gene, plays a
major role in 3?-to-5? exonucleolytic degradation of decay in-
termediates (16, 32, 42). In addition to PNPase, three other B.
subtilis 3?-to-5? exoribonucleases, RNase PH, RNase R, and
YhaM, can participate in mRNA decay (32). Recently, the role
of the RNase J enzymes (19) in B. subtilis mRNA turnover has
become apparent. While the RNase J enzymes were initially
purified on the basis of their endoribonuclease activity, it was
shown subsequently that the essential enzyme RNase J1 also
has 5?-to-3? exoribonuclease activity (29), which is inhibited by
a 5?-triphosphate end (14, 15). RNase J1 has been shown to be
involved in decay and processing of a number of specific RNAs
(2, 6, 14, 19, 45), and a transcriptome analysis demonstrated
that hundreds of mRNAs had increased half-lives in a strain
that had reduced levels of RNase J1 and in which the nones-
sential enzyme RNase J2 was deleted (27).
Other endonucleases of B. subtilis that have been characterized
to some extent are Bs-RNase III, RNase M5, RNase P, RNase Z,
be required for decay of an mRNA. Very recently, the product of
the essential gene ymdA (21) was renamed RNase Y by Com-
michau and colleagues, based on its association with other RNA-
processing enzymes and its apparent involvement in processing of
the gapA operon mRNA (7). Shahbabian and colleagues showed
that a strain in which RNase Y was depleted showed a significant
increase in global mRNA half-life, suggesting that this enzyme
plays a key role in mRNA turnover (38).
Previously, we used 5?-proximal oligonucleotide probes to
analyze the steady-state decay pattern of a number of small
monocistronic mRNAs by comparing the patterns detected by
5?-proximal probes in wild-type and pnpA strains (32). In each
of seven cases, prominent decay intermediates were observed
in the pnpA strain but not in the wild-type strain. One of the
mRNAs was the rpsO mRNA, a 388-nucleotide (nt) mRNA
that encodes ribosomal protein S15 (Fig. 1). The 3? ends of the
prominent rpsO decay intermediates detected in the pnpA
strain were mapped to the downstream side of predicted RNA
* Corresponding author. Mailing address: Department of Pharma-
cology and Systems Therapeutics, Box 1603, Mount Sinai School of
Medicine of New York University, New York, NY 10029. Phone: (212)
241-5628. Fax: (212) 996-7214. E-mail: firstname.lastname@example.org.
?Published ahead of print on 23 April 2010.
structures. We suggested that the accumulation of these inter-
mediates in the strains missing PNPase was the result of en-
donuclease cleavage downstream of the structure, followed by
3?-to-5? exonuclease degradation up to the 3? side of the struc-
ture. In strains containing PNPase, decay intermediates were
not readily detected since PNPase could degrade RNA struc-
tures. In the current study, we used rpsO mRNA to obtain data
supporting the endonucleolytic nature of decay initiation, and
we obtained evidence that RNase Y is the decay-initiating
endonuclease for rpsO mRNA.
MATERIALS AND METHODS
Bacterial strains. The triple and quadruple B. subtilis exoribonuclease mutant
strains used in this study were derivatives of parent strain BG1 (trpC2 thr-5),
which was designated the “wild-type” strain. Construction of exoribonuclease
mutant strains has been described previously (32). The conditional endonuclease
mutant strains used were RNase J1 (2), RNase P (43), RNase Z (33), and RNase
Y (24) mutants. These strains contained plasmid pMAP65 (34), which provided
additional copies of the lac repressor. B. subtilis strains were transformed as
described previously (18).
rpsO transcription under pbaccontrol. The pbac-rpsO construct was assembled
as follows. A 90-bp fragment of the lia operon promoter, including the LiaR
binding sites (28), was amplified by PCR using an upstream oligonucleotide that
included an MfeI restriction site. The rpsO transcriptional unit was amplified by
PCR using an upstream primer that contained a 7-nt sequence complementary to
the beginning of the downstream primer used for lia promoter region amplifi-
cation and a downstream primer that included a HindIII site after the rpsO
transcription terminator. The two PCR amplicons were annealed and were
amplified using the 5? lia promoter primer and the 3? rpsO primer. The resulting
product was digested with MfeI and HindIII and cloned into the EcoRI and
HindIII sites of plasmid pDR67-Pm, a derivative of the amyE integration plas-
mid pDR67 (22) in which the chloramphenicol resistance gene is replaced by a
phleomycin resistance gene (D. H. Bechhofer, unpublished data).
RNA analysis. RNA was isolated by hot phenol extraction from B. subtilis
cultures grown in minimal medium containing Spizizen salts with 0.5% glucose,
0.1% Casamino Acids, 0.001% yeast extract, 50 ?g/ml tryptophan, 50 ?g/ml
threonine, and 1 mM MgSO4, as described previously (12). All strains were
grown to the late logarithmic growth stage (100 Klett units, as determined using
a no. 54 green filter), except for the quadruple 3? exoribonuclease mutant, which
was grown to a density of 50 Klett units. For the experiment whose results are
shown in Fig. 3D, strains were grown in 2? YT medium containing 1% yeast
extract, 2% tryptone, and 1% NaCl to an optical density at 600 nm (OD600) of
0.6. Expression of RNase J1 and RNase Y in the conditional mutant strains was
induced with 1 mM isopropyl-?-D-thiogalactopyranoside (IPTG). For induction
of pbactranscription, bacitracin was added to a final concentration of 50 ?g/ml
when strains had grown to a density of 75 Klett units. Northern blot analysis of
RNA separated on 6% (see Fig. 2, 4, and 5) or 9% (see Fig. 3) denaturing
polyacrylamide gels was performed as previously described (20). 5?-End-labeled
oligonucleotide probes were prepared using T4 polynucleotide kinase and
[?-32P]ATP. To control for RNA loading, membranes were stripped and probed
for 5S rRNA, as described previously (39).
Data analysis. Quantitation of radioactivity in bands on Northern blots was
performed with a Storm 860 PhosphorImager instrument (Molecular Dynamics)
or a Typhoon TRIO variable-mode imager (GE Healthcare). The rpsO mRNA
half-life was determined by a linear regression analysis of the percentage of RNA
remaining versus time. Half-life data were obtained only from experiments in
which the R2value was greater than 0.9. Wild-type and mutant RNA half-lives
were compared using a two-sample t test to obtain P values. A P value of ?0.05
was considered significant.
Half-life of rpsO mRNA in exonuclease mutants. Experi-
ments were performed to determine whether the half-life of
rpsO mRNA is affected by a deficiency of the known B. subtilis
3?-to-5? exoribonucleases PNPase, RNase R, RNase PH, and
YhaM. The chemical half-life of full-length rpsO mRNA was
measured for strains that were deficient in three of the four
exonucleases or in all four exonucleases. The reasoning was as
follows. If decay was initiated by endonucleolytic cleavage,
then the half-life of the full-length mRNA should not be af-
fected by the absence of any of the known exonucleolytic ac-
tivities. The effect of the exonuclease deficiency would be pri-
marily on the fate of decay intermediates that are produced by
Total RNA was isolated from a B. subtilis wild-type strain
and from exonuclease mutant strains at different times after
addition of rifampin. Decay of rpsO mRNA was examined by
Northern blot analysis, using a 5?-end-labeled oligonucleotide
probe that was complementary to the translation initiation
region (nt 75 to 100) of the rpsO message (5?-proximal probe)
(Fig. 1). The Northern blots are shown in Fig. 2, and the
half-life data are shown in Table 1. The half-life of rpsO
mRNA in the wild-type strain was 3.2 min, and no prominent
decay intermediates were detected (Fig. 2A). The rpsO mRNA
half-life was slightly (but not significantly) longer (3.9 min) in
the strain that contained PNPase but not the other three exo-
ribonucleases (Table 1). As observed for the wild-type strain,
prominent decay intermediates were not detected (Fig. 2B).
We hypothesize that of the 3? exonuclease activities present in
B. subtilis, PNPase is the dominant activity and is able to
degrade past strong secondary structures and eliminate decay
intermediates (32). In the strain containing only RNase PH,
many decay intermediates were detected, some of them in
quantities that exceeded the amount of full-length mRNA
(e.g., the “180-nt” RNA) (Fig. 2C), and these decay interme-
diates were stable throughout the course of the experiment.
The same pattern was obtained when a 5?-terminal probe that
was complementary to nt 1 to 24 of rpsO mRNA was used (Fig.
1) (data not shown). Thus, the 5? end of these decay interme-
diates is likely to be at the transcription start site (TSS). De-
spite the massive accumulation of decay intermediates, the
half-life of full-length rpsO mRNA in the strain containing only
RNase PH was 4.3 min, which was not significantly different
from the half-life in the strain containing only PNPase (Table
1). The strain that contained only YhaM showed a pattern of
decay intermediates similar to that shown by the strain con-
taining only RNase PH (Fig. 2D), and the half-life was longer
(5.8 min) (Table 1). In the strain containing only RNase R, a
slightly different pattern of decay intermediates was detected
(Fig. 2E), but the half-life of full-length rpsO mRNA was also
FIG. 1. Schematic diagram of the rpsO transcript, showing the lo-
cations of the Shine-Dalgarno sequence (filled rectangle), start and
stop codons, five predicted stem-loop structures which appear to block
3? exonuclease processivity (32), and 3? transcription terminator
(3?TT). The relative predicted strengths of the stem-loop structures
are indicated roughly by the sizes of the stems. The locations of com-
plementary probes used in Northern blot experiments to detect 5?-end-
containing decay intermediates are also indicated.
3280YAO AND BECHHOFER J. BACTERIOL.
5.8 min. Finally, in the strain that was deficient for all four of
the known 3?-to-5? exoribonucleases, the half-life of full-length
rpsO mRNA was 4.9 min (Fig. 2F and Table 1). The half-lives
observed did not correlate with the previously measured dou-
bling times of the mutant strains (32). For example, the qua-
druple mutant has a doubling time that is 1.5 to 2 times longer
than the doubling times of the triple mutants, yet the rpsO
mRNA half-life was shorter in the quadruple mutant. These
results indicated that 3? exonuclease activity has a minor role in
determining the rpsO mRNA half-life. Endonuclease cleavage
was presumably the major determinant of the mRNA half-life.
Decay intermediates containing the 3? end. Endonuclease
cleavage in the body of the rpsO message should generate
upstream fragments containing the rpsO mRNA 5? end (easily
detected by the 5?-proximal probe in the PNPase-deficient
mutants, as shown in Fig. 2) and downstream fragments con-
taining the rpsO mRNA 3? end. The stability of the down-
stream fragment should depend on its susceptibility to addi-
tional cleavage with endonucleases or to the 5?-to-3?
exonuclease activity of RNase J1. Presumably, the presence of
the transcription terminator structure at the 3? end of the
downstream fragment protects against 3?-to-5? exonucleolytic
decay. We sought to detect such downstream fragments by
using 11 overlapping oligonucleotide probes, each 36 nt long,
that were complementary to sequences starting from the rpsO
transcription terminator past the midpoint of the coding se-
quence (CDS) (Fig. 3A). These oligonucleotides were used in
Northern blot analyses of RNA isolated from the wild-type
strain (Fig. 3B). The blots were exposed for much longer times
than those shown in Fig. 2. Figure 3B shows that fragments
that contained the rpsO mRNA 3? end could be detected,
although the amounts were much smaller than the amounts of
full-length mRNA. Multiple species were detected, and the
clearest groups of bands were designated b to j, from the
largest to the smallest (the full-length rpsO mRNA band was
designated band “a”). From the data, it was clear that the 5?
ends of the smallest fragments were closest to the 3? end of
rpsO mRNA, since they could be detected only by the 3?-
proximal probes. The 5? ends of the larger fragments were
located increasingly further upstream, since they could also be
detected by probes complementary to sequences further up-
stream in the CDS. If we assume that comigrating bands ob-
served with the different probes represent the same RNA frag-
ments, then the patterns of the Northern blots demonstrated
that all of the RNAs detected by the probes contained the 3?
end of rpsO mRNA. While there were not sufficient amounts of
these RNAs to map their 5? ends precisely, the extents of the
RNA fragments could be approximated, based on size markers
run in parallel and assuming that the fragments shared the
same 3? end. These fragments are shown schematically in Fig.
3A. Interestingly, the fragments appear to cluster in the 3?-
proximal half of the transcript. We hypothesize that these
RNA fragments resulted from one or more endonuclease
cleavages downstream of the strong stem-loop near nt 180 and
subsequent 5?-to-3? processing. The fragments were detectable
because they were protected by the transcription terminator
structure, but, unlike the 5?-end-containing fragments (Fig. 2),
FIG. 2. Northern blot analysis of rpsO mRNA decay in 3?-to-5?
exoribonuclease mutants. The genotypes for the four known 3? exori-
bonucleases are indicated below the panels. The numbers above the
lanes are the times (in minutes) after rifampin addition. The probe was
a 5?-end-labeled oligonucleotide complementary to the rpsO transla-
tion initiation region (nt 75 to 100) (Fig. 1). The position of full-length
rpsO mRNA is indicated by the arrow on the left in each panel. The
position of the prominent 180-nt decay intermediate is indicated on
the right. The quantity of total RNA in each lane was corrected using
the amount of RNA detected by a 5S rRNA-specific oligonucleotide
probe, as shown at the bottom in each panel.
TABLE 1. RNase mutants and rpsO half-lives
rpsO half-life (min)
(mean ? SD)c
Wild type PNPase, RNase PH, YhaM, RNase RNAe
3.24 ? 0.47 NA
rnr::Tc rph?Sp yhaM::Pm
pnpA::Cm rnr::Tc yhaM::Pm
pnpA::Km rnr::Tc rph?Sp
pnpA::Km rph?Sp yhaM::Pm
3.94 ? 0.39
4.30 ? 0.73
5.84 ? 0.82
5.76 ? 0.65
QuadrupleNonepnpA::Cm rnr::Tc rph?Sp yhaM::Pm 4.85 ? 0.15
aThe presence of the four known 3?-to-5? exoribonucleases is indicated.
bThe resistance markers were chloramphenicol (Cm), kanamycin (Km), phleomycin (Pm), spectinomycin (Sp), and tetracycline (Tc) resistance.
cThe data are the results of three experiments.
dTo determine P values, the half-lives for the mutant strains were compared to the half-life for the wild type.
eNA, not applicable.
VOL. 192, 2010RNase Y-MEDIATED INITIATION OF mRNA DECAY 3281
they were unstable because they were susceptible to further
endonucleolytic or 5? exonucleolytic attack.
Pattern of 3?-end-containing fragments in exoribonuclease
mutants. A considerable difference in the pattern of 5?-end-
containing decay intermediates was observed between strains
that contained PNPase and strains that did not contain PNPase
(Fig. 2, compare panels A and B and panels C to F). According
to our model, these decay intermediates arose by endonuclease
cleavage in the body of the message, followed by 3?-to-5? exo-
nucleolytic decay up to the 3? side of RNA structures, which
blocked 3? exonucleases other than PNPase (32). On the other
hand, we predicted that the pattern of 3?-end-containing decay
fragments (Fig. 3B), which were the downstream fragments
resulting from endonuclease cleavage, should not be affected
significantly by the type of 3? exonuclease activity present in the
cell. This prediction was tested directly by performing a North-
ern blot analysis of 3?-end-containing fragments in the various
triple exonuclease mutant strains, using 3?-terminal probe 1
(Fig. 3C). The results showed that the patterns of 3?-end-
containing fragments in the wild-type and RNase mutant
strains, although not identical, were similar in terms of the
sizes and amounts of RNA fragments.
Levels of 3?-end-containing fragments in RNase J mutant
strains. Although 3?-end-containing fragments were detect-
able in the wild-type strain, their abundance was relatively low.
We tested whether RNase J1 or RNase J2 was responsible for
degradation of these fragments by repeating the Northern blot
analysis using probe 1 with RNA isolated from RNase J mu-
tant strains. In the RNase J1 mutant strain rnjA expression is
under the control of an IPTG-inducible promoter. When
grown in the absence of IPTG, the RNase J1 mutant strain
contains a much lower level of enzyme, while growth in the
presence of IPTG results in a ?5-fold-lower level of enzyme
(10). As Fig. 3D shows, the intensity of the steady-state pattern
of small fragments in the RNase J1 mutant strain grown with
IPTG was somewhat greater than that in the wild-type strain
but the intensity was much greater when the organism was
grown without IPTG. Deletion of the RNase J2 gene did not
have a clear effect on the decay intermediate pattern. (In this
experiment, the amount of full-length RNA in the RNase J2
mutant lane was about 20% greater than the amount in the
wild-type lane. Hence, the lower bands in the RNase J2 mutant
lane are more visible than those in the wild-type lane, but the
patterns are the same.) We concluded that RNase J1 is
FIG. 3. Detection of 3?-end-containing mRNA decay fragments. (A) Linear diagram of rpsO transcript. The box representing the CDS
corresponds to the interval between the AUG start codon and the UAA stop codon shown in Fig. 1. The region of complementarity for each of
the 11 overlapping 3?-proximal probes (probes 1 to 11) used in the Northern blots in panel B is indicated. The approximate extents of
3?-end-containing mRNA decay fragments, designated using the letters used in panel B, are indicated below the probes. Fragment a is full-length
rpsO mRNA, and the stem-loop that ends at nt 172 (see Fig. 1) and that gives rise to the “180-nt” decay intermediate is shown for reference. The
diagrams for RNA fragments b to j represent not single RNAs but groups of RNAs with 5? endpoints that are close to each other. (B) Northern
blot analysis of 3?-end-containing rpsO decay fragments in the wild-type strain. The probe used is indicated below each blot. Groups of fragments
are labeled b to j to the right of each lane containing total RNA. The marker lanes (lanes M) contained 5?-end-labeled fragments of a TaqI digest
of plasmid pSE420 (3). The numbers on the left indicate molecular sizes (in nucleotides). (C) Northern blot analysis of 3?-end-containing fragments
in triple RNase mutants, using probe 1. The wild type (wt) contained all four known 3?-to-5? exoribonucleases. The genotypes for the four known
3? exoribonucleases are indicated on the right. For an explanation of lane M see above. (D) Northern blot analysis of 3?-end-containing fragments
in RNase J1 and RNase J2 mutant strains, using probe 1. The RNase J1 conditional mutant was grown in the presence (?) or absence (?) of IPTG,
as indicated. For an explanation of lane M see above.
3282 YAO AND BECHHOFERJ. BACTERIOL.
responsible for turnover of the downstream fragments that
arise by endonuclease cleavage.
rpsO mRNA half-life in endonuclease mutant strains. At the
time that this study of rpsO mRNA decay was begun, RNase J1
was the only endonuclease of B. subtilis known to be involved
in initiation of mRNA decay. We thus thought that RNase J1
could be responsible for the endonucleolytic cleavage that was
suggested by the analysis of rpsO mRNA decay intermediates.
Northern blot analysis was used to measure the rpsO mRNA
half-life in the RNase J1 conditional mutant strain grown in
the presence of 1 mM IPTG. The decrease in the RNase J1
level in the conditional mutant strain, even when IPTG is
present, is sufficient to detect changes in the half-lives of RNAs
whose decay is RNase J1 dependent (10, 13, 44). We found,
however, that the half-life of rpsO mRNA in the RNase J1
mutant was 3.9 min (data not shown), which is not significantly
different from the 3.2-min half-life in the wild type. The half-
life in an RNase J1 mutant strain in which the RNase J2 gene
was deleted was similar (3.8 min). More recent reports have
identified RNase Y as a potentially major player in RNA decay
and processing in B. subtilis (see Discussion). We therefore
measured the half-life of rpsO mRNA in the RNase Y condi-
tional mutant grown in the presence of 1 mM IPTG. The
growth rate of the RNase Y mutant strain under these condi-
tions was only slightly lower than that of the wild type (data not
shown). Northern blot analysis of rpsO mRNA decay in the
wild-type and RNase Y mutant strains after addition of ri-
fampin showed that there was a 2.3-fold increase in the mRNA
half-life (Fig. 4).
System to monitor the appearance of decay intermediates
over time. The prominent rpsO mRNA decay intermediates
observed at steady state in the PNPase-deficient strains (Fig. 2)
suggested that we could use an inducible system to follow
accumulation of these fragments over time in order to confirm
the involvement of a particular endonuclease in initiation of
decay. Several problems were encountered when we per-
formed time course analyses using the conventional IPTG-
inducible or xylose-inducible promoters. First, since the rpsO
5?-end-containing decay intermediates were extremely stable
(Fig. 2), we needed an inducible transcription system that was
not “leaky,” as ongoing low-level transcription in the absence
of inducer would lead to steady-state accumulation of RNA
fragments. The IPTG- and xylose-inducible systems, which are
negatively regulated by repressors, were somewhat leaky (data
not shown). Second, examination of the appearance of decay
intermediates requires an induction system that scales up to
full induction over a reasonably long period of time. We found
that full induction occurred over an extremely short time pe-
riod when IPTG- and xylose-inducible systems were used (data
not shown). Third, regulation of the commonly used inducible
promoters depends on the presence of an operator sequence
located downstream of the TSS. This means that the induced
transcript contains the cognate operator sequence, which gives
a 5?-proximal sequence very different from the native tran-
script. This may be problematic for studying mRNA decay that
could be 5? end dependent.
To avoid some of these issues, we created a new inducible
system that relies on the bacitracin-inducible promoter of the
lia operon (28). This promoter is induced positively by subin-
hibitory concentrations of bacitracin, and we designated it the
“pbac” promoter. A 90-bp fragment encompasses the promoter
and the upstream regulatory region at which the transcrip-
tional activator, LiaR, binds when bacitracin is present (28). A
construct in which the pbacpromoter fragment was located so
that transcription started at the rpsO TSS was integrated at the
amyE locus to obtain “pbac-rpsO mRNA.” To differentiate be-
tween native rpsO mRNA and pbac-rpsO mRNA, nt 8 to 16 of
the rpsO sequence (UAAAACCAU) were changed to the
complementary sequence. B. subtilis rpsO mRNA begins with a
leader region (Fig. 1), which includes a predicted pseudoknot
structure (41) that is thought to be involved in translational
autoregulation (35, 36). However, the pseudoknot structure
begins at nt 30, and we assumed that changing nt 8 to 16 would
not affect decay characteristics of the message. In these exper-
iments, we used a bacitracin concentration of 50 ?g/ml (equiv-
alent to 3.5 U/ml), which results in full induction but does not
significantly affect bacterial growth (31).
The results of a Northern blot analysis of the induction
kinetics of pbac-rpsO mRNA in the pnpA strain are shown in
Fig. 5A. The probe was directed to the 5? end of pbac-rpsO
mRNA. Before addition of bacitracin (Fig. 5A, lane B) no
signal was detected, demonstrating that the probe does not
detect native rpsO mRNA. The pbac-rpsO mRNA was detected
faintly at 1 min, and the level increased over time; almost full
induction occurred at around 15 min (Fig. 5B). At this time,
the prominent 180-nt decay intermediate was visible, suggest-
ing that the nature of the promoter and the change in the
5?-proximal sequence do not affect mRNA processing.
Induction of pbac-rpsO mRNA in endonuclease mutants. A
time course analysis of the accumulation of full-length pbac-
rpsO mRNA and decay intermediates was performed using
several endonuclease mutant backgrounds. There was no dif-
ference between the single-mutant pnpA strain and the pnpA
strain that had decreased levels of RNase J1 and no RNase J2
(Fig. 5B). This finding correlated with the similar half-lives of
rpsO in the wild-type and RNase J1 mutant strains, as de-
scribed above. Similarly, the bacitracin-induced time course
was not affected in strains with reduced levels of RNase P and
RNase Z (data not shown). However, the results obtained with
the RNase Y mutant strain were informative. At the earliest
time point (0.5 min), pbac-rpsO mRNA was virtually undetect-
able in the pnpA strain but was clearly present in the double
mutant strain with reduced RNase Y (Fig. 5C). Over time,
compared to full-length pbac-rpsO mRNA, the amount of the
180-nt decay intermediate that accumulated increased much
more in the pnpA strain than in the double-mutant strain with
FIG. 4. Northern blot analysis of rpsO mRNA decay in wild-type
and RNase Y mutant strains. The probe was the 5?-proximal probe
(Fig. 1). The numbers above the lanes indicate the times (in minutes)
after rifampin addition. The measured half-life (t1/2) (average ? stan-
dard deviation of three experiments) is indicated below each blot. For
an explanation of the marker lane on the left see the legend to Fig. 3.
VOL. 192, 2010 RNase Y-MEDIATED INITIATION OF mRNA DECAY 3283
reduced RNase Y (Fig. 5D). These results are consistent with
initiation of decay by RNase Y cleavage. One other prominent
RNA, which was about 270 nt long, was detected, but quanti-
tative analysis of this RNA showed that it did not accumulate
compared to the full-length RNA (Fig. 5D).
The analysis of rpsO mRNA half-life in 3? exonuclease mu-
tants (Table 1 and Fig. 2) indicated that 3?-to-5? exoribonucle-
ases have a minor role in determining this mRNA half-life. We
observed slight but not statistically significant increases in the
rpsO mRNA half-life in strains containing only PNPase or only
RNase PH and ?2-fold increases in the rpsO mRNA half-life
in strains containing only YhaM, only RNase R, or none of the
known 3? exonucleases. These results suggested that 3? exo-
nucleases have some effect on the half-life of full-length rpsO
mRNA, but endonuclease cleavage was likely more important
for initiation of decay. We have no good explanation at present
for the greater increase in the rpsO mRNA half-life in the
strains containing only YhaM or RNase R, especially since the
increase was greater than that in the strain containing none of
the four known exoribonucleases (Table 1). Perhaps particular
perturbations in the exoribonuclease complement of the cell
indirectly affect endoribonuclease activity; recent evidence for
a putative B. subtilis degradosome complex that includes
PNPase, RNase J1, and RNase Y (7) may be indicative of
other interactions between RNases.
Endonuclease cleavage in the rpsO mRNA decay pathway
was also inferred from the detection of multiple RNA frag-
ments that were different sizes but all contained the 3? end
(Fig. 3B). We found that the patterns were similar for the
wild-type and exoribonuclease mutant strains examined (Fig.
3C), despite the enormous differences between these strains
when the patterns detected with the 5?-proximal probe were
examined (Fig. 2). The contrast between the complete lack of
5?-proximal decay intermediates in the strain containing only
PNPase (Fig. 2B) and the presence of 3?-proximal decay inter-
mediates in the same strain (Fig. 3C, lane A) is particularly
striking and is consistent with initiation of decay by endonu-
clease cleavage. The 3?-terminal fragments may be direct prod-
ucts of endonuclease cleavage, or they may be secondary prod-
ucts of RNase J1 5? exonuclease activity that proceeds from the
5? end(s) generated by endonuclease cleavage. The low level of
3?-end-containing fragments in the wild-type strain likely oc-
curs because RNase J1 is capable of degrading through the
secondary structure in the 5?-to-3? direction (14). Indeed, de-
pleting the cell severely (without IPTG) or moderately (with
IPTG) of RNase J1 resulted in increased intensity of the 3?-
end-containing decay intermediates (Fig. 3D).
We found in assays of mRNA half-lives and of accumulation
of full-length RNA and decay intermediates that RNase J1 was
not responsible for determining the stability of rpsO mRNA.
Although RNase Y, the product of the ymdA gene, was sus-
pected long ago of being an RNase (1, 9), only in the last year
have data on the role of RNase Y in RNA processing been
described. Meinken and colleagues obtained evidence that
cleavage of gapA operon mRNA at a particular site (30) was
FIG. 5. Time course of pbac-rpsO mRNA accumulation. (A) Induction of pbac-rpsO transcription. The positions of full-length (FL) pbac-rpsO and
a prominent decay intermediate (180 nt) are indicated on the right. The numbers above the lanes indicate the times (in minutes) after addition
of bacitracin. The “zero” time point in panels A to C is actually the interval between addition of bacitracin and removal and processing of the first
aliquot, which was about 30 s. Lane B contained RNA isolated before addition of bacitracin. For an explanation of the marker lane (lane M) see
the legend to Fig. 3. (B) Accumulation of decay intermediates in the pnpA strain and in the pnpA strain with a reduced level of RNase J1 and no
RNase J2. (C) Appearance of decay intermediates in the pnpA strain and the pnpA strain with a reduced level of RNase Y. (D) Ratios of the 180-nt
decay intermediate (solid lines) and the 270-nt decay intermediate (dashed lines) to full-length pbac-rpsO in the pnpA strain (diamonds) and the
pnpA Pspac-rny double mutant strain (squares). Error bars are shown only for the 180-nt decay intermediate. The RNase genotypes and fragment
sizes (in nucleotides, in parentheses) are indicated on the right.
3284YAO AND BECHHOFERJ. BACTERIOL.
due to RNase Y (7). Shahbabian and colleagues demonstrated
that cleavage of the yitJ riboswitch RNA, as well as other
S-adenosylmethionine-dependent riboswitches, could be at-
tributed to RNase Y (38). They also found that depletion of
RNase Y resulted in an increase in the half-life of bulk mRNA.
Thus, we turned our attention to RNase Y and showed, for the
first time, that RNase Y has an effect on decay of a specific B.
subtilis mRNA. We observed a ?2-fold increase in the rpsO
mRNA half-life in the RNase Y conditional mutant (Fig. 4).
This suggests that there is a strong dependence on RNase Y
for initiation of decay, since we assumed that the RNase Y
conditional mutant grown with 1 mM IPTG contains a signif-
icant level of the enzyme. We have not proven that RNase Y
acts directly on rpsO mRNA, which would require in vitro tests,
and it is possible that a deficiency in RNase Y has indirect
effects on initiation of mRNA decay. Nevertheless, the sim-
plest interpretation of our results is that RNase Y cleaves rpsO
mRNA, and for the discussion below we assume that this is the
The pbacsystem was useful for demonstrating faster accu-
mulation of full-length rpsO mRNA in the RNase Y mutant
strain (Fig. 5C, zero-time lane), presumably because initiation
of decay was slower due to the lower level of RNase Y. We
hypothesize that the 180-nt RNA results from endonuclease
cleavage followed by 3? exonuclease activity up to the strong
stem-loop structure that ends at nt 172. Thus, if RNase Y
cleavage is required to generate this RNA, accumulation of the
RNA should be slower in the RNase Y mutant, and this was
indeed the case (Fig. 5C and 5D). Other bands, in addition to
the full-length and 180-nt bands, were detected (Fig. 5C).
Some of these bands were faint and were present throughout
the time course, and these bands likely represent nonspecific
hybridization. We speculate that the band at around 270 nt
may be a prematurely terminated transcription product or a
processing product of a different RNase acting on full-length
From the current analysis of rpsO mRNA decay intermedi-
ates, it was not clear if RNase Y cleaves once or several times
in the body of the message. Even a single endonuclease cleav-
age could give rise to numerous decay intermediates due to
subsequent 3?-to-5? exonuclease and 5?-to-3? exonuclease ac-
tivities and hindrance of these activities by RNA structure. In
any event, the results obtained to date allow construction of a
preliminary model for the complete turnover of rpsO mRNA,
which begins with endonuclease cleavage by RNase Y and is
completed by the 3? exonuclease activity of PNPase on up-
stream products and the 5? exonuclease activity of RNase J1 on
downstream products (Fig. 6). As RNase Y is essential, we
expect that RNase Y catalyzes decay-initiating cleavage of
many B. subtilis mRNAs.
Unlike RNase J1, which has robust endonuclease activity
with 5? triphosphorylated substrates (14, 15), RNase Y endo-
nuclease activity is sensitive to the nature of the 5? end, and the
in vitro activity on RNA with a 5? monophosphorylated end is
significantly higher (38). If this is also true of RNase Y activity
in vivo, it has major consequences for models of RNase Y-
dependent initiation of decay. Figure 2 shows that decay inter-
mediates detected in the PNPase-deficient strains were ex-
tremely stable, and their intensities did not decrease
throughout the experiment. This suggests that degradation by
RNase J1 5? exonucleolytic activity from the native 5? end did
not occur, even though the data in Fig. 3D indicate that the
same 5? exonuclease activity degraded the 3?-end-containing
fragments. This suggests that the 5? triphosphate group of the
initial rpsO transcription product is not removed, making 5?-
end-containing decay intermediates resistant to RNase J1 5?
exonucleolytic decay. However, if this is the case and if RNase
Y is sensitive to the 5? triphosphate end, then it is hard to
understand how internal cleavage by RNase Y resulting in a
relatively short half-life (3.2 min) occurs. Much work is needed
to understand the basis of endonucleolytic cleavage by RNase
J1 and RNase Y and to determine why particular messages are
subject to one of the activities or perhaps both activities.
This work was supported by Public Health Service grant GM-48804
from the National Institutes of Health to D.H.B.
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