(Received for publication, October 18, 1996, and in revised form, January 7, 1997)
From The Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853-1801
3-Untranslated region stem-loop structures are
major determinants of chloroplast mRNA stability. The 3
stem-loop
region of spinach petD precursor mRNA (pre-mRNA), a
chloroplast gene encoding subunit IV of the cytochrome
b6·f complex, forms a stable RNA-protein complex in vitro with chloroplast stem-loop
binding proteins (CSPs) of 55, 41, and 29 kDa. We have previously
purified CSP41 and cloned the corresponding cDNA. In
vitro studies demonstrated that CSP41 is a bifunctional protein
that displays both endoribonuclease and RNA-binding activities. In this
work, the RNase activity of CSP41 is further characterized using the
bacterially expressed protein. Our data show that CSP41 cleaves both
single-stranded and double-stranded RNAs but not DNA. However, it
exhibits a preference for stem-loop-containing RNAs. When the
3
-untranslated region of petD pre-mRNA is provided as
a substrate, CSP41 specifically cleaves it within the stem-loop region,
implying that CSP41 has an important role in the control of
petD mRNA stability. Our data also show that the
sequence-specific RNA-binding activity of CSP41 affects the rate, but
not the specificity, of its RNase activity, suggesting that CSP41 is
probably involved in other events of chloroplast RNA metabolism in
addition to RNA degradation. By analyzing C-terminal deletions of
CSP41, the RNase domain was located between amino acid residues 73 and
191.
Most chloroplast precursor mRNAs (pre-mRNAs) and mature
mRNAs contain an inverted repeat sequence in their 3-untranslated region (UTR)1 that can fold into a stable
stem-loop structure. Both in vitro and in vivo
studies have demonstrated that these 3
stem-loop structures are
required for correct 3
processing of pre-mRNAs and for
stabilization of mature mRNAs, by impeding processive 3
to 5
exonucleolytic degradation. In the absence of a stable secondary
structure, or by readthrough of the stem-loop structure, mRNA
becomes heterogeneous and generally unstable (1-7). Therefore, the 3
stem-loop structure has a primary role in controlling chloroplast mRNA accumulation through pre-mRNA processing and the
protection of upstream sequences. Removal of the 3
stem-loop
structure, therefore, is likely to be a key regulatory step in
chloroplast mRNA degradation.
Bacterial pre- and mature mRNAs are another major group of
mRNAs containing 3-inverted repeats. The 3
stem-loop structures of bacterial mRNAs function in the processing of pre-mRNAs and the stabilization of mature mRNAs by a mechanism similar to that found in chloroplasts (8). Extensive studies have shown that at least
three groups of proteins are involved in the control of 3
to 5
degradation of bacterial mRNAs: 1) endoribonucleases, such as RNase
E and RNase III, which cleave the 3
-UTR within or upstream of the
stem-loop structure to overcome the secondary structure barrier; 2)
exoribonucleases, such as RNase II and polynucleotide phosphorylase,
which bind to the free 3
end created by the endoribonucleases and
degrade mRNA in the 3
to 5
direction; and 3) stem-loop-binding proteins, which modulate degradation by interacting with RNA and/or RNases (8-11). Each of these classes of protein can be found within a
single multiprotein complex, termed the "degradosome" (12-14).
Several reports have documented proteins that may be involved in the
maturation and/or 3 to 5
degradation of chloroplast mRNA (6, 15,
16). It was recently reported that a multiprotein complex containing a
polynucleotide phosphorylase homologue and a putative RNase E-like
protein could function in spinach chloroplast mRNA 3
processing
and degradation (17). We have previously identified a stable
stem-loop-protein complex in the 3
-UTR of spinach petD
pre-mRNA, a chloroplast gene encoding subunit IV of the cytochrome
b6·f complex (18). Complex
formation requires not only the stem-loop but also an AU-rich element
(box II) immediately downstream of the stem-loop. Protein components of
the complex include chloroplast stem-loop binding proteins (CSPs) of
55, 41, and 29 kDa. CSP41 was purified and the corresponding nuclear
gene was cloned (19). Bacterially expressed and purified CSP41
displayed both endoribonuclease activity and sequence-specific
RNA-binding activity. A detailed characterization of the RNA-binding
activity indicated that CSP41 can bind to the stem-loop and to box II
and may interact with the other CSPs. CSP41's dual functions raised the question of how these activities were interrelated and regulated. Here, we report a detailed biochemical characterization of CSP41 endoribonuclease activity. Of particular significance is that CSP41
primarily cleaves the 3
-UTR of petD mRNA within the
stem-loop structure, consistent with a key role in the control of
chloroplast mRNA stability.
Four
stem-loop-containing RNAs were used. As shown in Fig. 1, the 208-nt
petD50 wild-type (WT) and box II mutant (BIIa) RNAs include 84 nucleotides of the 3
end of the petD coding region and 107 nucleotides of the petD pre-mRNA 3
-UTR, encompassing the stem-loop structure and its upstream and downstream AU-rich elements (box I and box II). The 176-nt psbA135 and 156-nt rbcL115 RNAs
contain vector sequences plus 135 nucleotides of the psbA pre-mRNA 3
-UTR and 115 nucleotides of the rbcL
pre-mRNA 3
-UTR, respectively, beginning immediately downstream of
the stop codons and encompassing their stem-loop structures. The
rbcL115 RNA also includes a box II-like AU-rich element. Both petD
50
RNAs were synthesized as described previously (4). For psbA135 and
rbcL115 RNAs, the corresponding DNAs were amplified from spinach total DNA by the polymerase chain reaction and cloned into pBluescript KS+
(Stratagene, La Jolla, CA) between the XbaI and
EcoRI sites. The resulting plasmids were linearized with
EcoRI and transcribed with T7 RNA polymerase in the presence
of [
-32P]UTP. For 5
-end labeling, nonradioactive
petD
50 RNA was dephosphorylated by alkaline phosphatase and labeled
with [
-32P]ATP and polynucleotide kinase (4). Before
use, all transcripts were purified from 6% denaturing polyacrylamide
gels.
To make uniformly labeled single- and double-stranded RNAs, pBluescript
SK+ was separately linearized with SacI and KpnI
and transcribed with T7 and T3 RNA polymerases, respectively, yielding a 118-nt RNA from the T7 promoter and a 121-nt RNA from the T3 promoter. These RNAs were gel-purified, mixed, boiled for 5 min, and
then cooled to room temperature. The resulting double-stranded RNA was
purified from an 8% native polyacrylamide gel. To make 5-end-labeled
single- and double-stranded DNAs, the polylinker fragments between the
NotI and KpnI sites and between the
SacI and KpnI sites were dephosphorylated and
then labeled with [
-32P]ATP. The two strands of the
labeled NotI-KpnI fragment were separated in a
6% denaturing polyacrylamide gel, and the longer strand (89 nt) was
recovered as 5
-end-labeled single-stranded DNA. The labeled 94-nt
double-stranded SacI-KpnI fragment was purified
through a 4-ml Sephadex G-25 spin column. An RNA ladder was generated
by alkaline hydrolysis of 30,000 cpm of 5
-32P-petD
50WT
RNA (15).
Plasmid pQE30-csp41 (19) was used to produce
N-terminal 6 × histidine-tagged wild-type CSP41 (HCSP41). Three 3
deletions of the csp41 gene were made by restriction enzyme
digestion and religation of the pQE30-csp41 plasmid.
pQE30-csp41
E was created by digestion with
EcoRV and SalI, pQE30-csp41
S with
SmaI, and pQE30-csp41
P with PstI.
These modified plasmids were used to produce the HCSP41 C-terminal
deletion mutants HCSP41
E (284 amino acids), HCSP41
S (191 amino
acids), and HCSP41
P (73 amino acids), respectively. HCSP41 and the
mutant proteins were prepared as described (19), except that to recover
both RNase and RNA-binding activities of HCSP41, refolding was carried
out in a buffer system containing 0.02 mM oxidized and 2 mM reduced glutathione for a minimum of 72 h. The
initial concentration of denatured protein was limited to less than 0.5 µg/ml. Homogeneous HCSP41 was obtained by purification on a
HiTrap-heparin FPLC cartridge (Pharmacia Biotech Inc.) (19), whereas
the mutant proteins were only purified through the Ni-NTA column. Using
the oligonucleotide ATCCCCTTCAATAGCAGGAGGTGCAGCTGTAGAATT, pQE30-csp41A126 was created by site-directed mutagenesis
(20). HCSP41A126 mutant protein was produced and prepared as described above. Protein profiles were examined in a Bio-Rad
mini-SDS-polyacrylamide gel electrophoresis system and visualized by
Coomassie Blue staining.
The 30-µl standard RNase assay and the 20-µl standard gel mobility shift assay were carried out as described previously (19), except that the both reaction mixtures had a pH of 7.5 instead of 7.9, and the RNase assay was carried out at 30 °C. To visualize RNA degradation and RNA binding, samples were subjected to denaturing or native polyacrylamide gel electrophoresis and then exposed to x-ray film. The quantification of both RNase and RNA-binding activities was accomplished using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
We have previously reported that spinach CSP41, a
nuclear-encoded chloroplast RNA-binding protein, can specifically bind
to the 3 stem-loop region of petD pre-mRNA.
Surprisingly, we found that highly purified CSP41 also exhibited robust
endoribonuclease activity, cleaving the 3
-UTR of spinach
petD pre-mRNA into multiple fragments of poly- and
oligoribonucleotides (19).
To further characterize the RNase activity of CSP41, we expressed the
N-terminal 6 × histidine-tagged CSP41 fusion protein (HCSP41) in
E. coli and purified it through a Ni-NTA column under denaturing conditions. HCSP41 was refolded under optimized conditions and further purified by heparin-FPLC (see "Experimental
Procedures"). This protocol yielded a preparation of homogeneous
HCSP41 with reproducible and robust endonuclease activity. Fig.
2A shows that when uniformly labeled 208-nt petD50WT RNA
(Fig. 1) was provided as substrate, HCSP41 cleaved it
into smaller fragments identical to those we have reported previously,
although in different relative amounts. In this standard assay, 0.4 µg of refolded HCSP41 cleaved approximately 90% of the full-length
substrate in 25 min.
Since petD50WT RNA contains a stable 46-nt stem-loop structure (see
Fig. 1), it was unclear whether the endonuclease activity of CSP41 was
specific for single-stranded or double-stranded RNA. It was also
unknown whether CSP41 could digest DNA, as other endonucleases do (21).
To test CSP41 specificity, uniformly labeled 121-nt single- and
116-base pair double-stranded RNAs and 5
-end-labeled 89-nt single- and
94-base pair double-stranded DNAs were provided as substrates under our
standard conditions. Fig. 2, B and
C, shows that HCSP41 can cleave both single- and
double-stranded RNAs, but neither single- nor double-stranded DNAs,
demonstrating that CSP41 is an RNA-specific endonuclease. However,
quantification of the ribonuclease activities using a PhosphorImager
revealed that only 39% of the single-stranded RNA and 36% of the
double-stranded RNA was shortened, a much lower amount than that seen
with petD
50WT RNA under the same conditions (Fig. 2A).
The higher degradation rate of petD
50WT RNA implies either that
petD
50WT RNA contains preferred target sequences or structures for
the RNase activity of CSP41 but the shorter RNA substrates do not or
that the sequence-specific binding of CSP41 to petD
50WT RNA enhances
its RNase activity (see below).
To characterize more completely the properties of the RNase activity,
we performed RNA degradation assays under a variety of conditions.
Table I summarizes the results, where we define the
initial cleavage of petD50WT RNA in 25 min under standard conditions
(Fig. 2A) as 1.0 unit of activity. The data show that 1)
CSP41 is active up to at least 55 °C and has a temperature optimum
between 37 and 45 °C; 2) the pH optimum is neutral; 3) high
concentrations of mono- or divalent cations, or a complete removal of
divalent cations by EDTA reduces or abolishes activity; and 4) ATP does
not affect RNase activity.
|
The RNA-binding activity of CSP41
requires an AU-rich element immediately downstream of the
petD 3 stem-loop region. Mutations in this element, such as
the BIIa mutation shown in Fig. 1, abrogate CSP41 binding (19). To
investigate whether the RNA-binding activity of CSP41 enhances the
RNase activity of CSP41 when petD
50 RNA is the substrate, we
utilized uniformly labeled petD
50WT, petD
50BIIa, psbA135, and
rbcL115 RNAs. As shown in Fig. 1, these RNAs contain parts of the
3
-UTRs of wild-type petD pre-mRNA, mutant
petD pre-mRNA, psbA pre-mRNA, and
rbcL pre-mRNA, respectively. Each of these RNAs contains
a 3
stem-loop structure and flanking sequences, but only petD
50 and
rbcL115 possess an immediately downstream AU-rich element.
To examine their interactions with CSP41, the RNAs were first used in a
gel mobility shift assay. As expected, in a standard assay containing
0.4 µg of HCSP41 and 4 fmol of RNA, HCSP41 formed a complex with
petD50WT RNA but not with either petD
50BIIa or psbA135 RNA, both
of which lack the downstream AU-rich element (Fig. 3).
HCSP41 also formed a complex with rbcL115 RNA, but the signal was much
weaker. This difference may be due to the one-nucleotide difference
between the AU-rich elements or to other sequence or structural
differences. In contrast, when used as substrates in standard RNase
assays, we found that each RNA was rapidly cleaved into smaller
fragments. Even in the relatively slow reactions, such as those with
psbA135 or rbcL115 RNAs, less than 20% of the precursor remained after
25 min, as shown in Fig. 4A. It is especially notable that HCSP41 cleaves petD
50WT and petD
50BIIa nearly
identically, although the protein binds stably only to petD
50WT RNA
under these conditions. When samples were taken over the 25-min time course, the profiles of petD
50WT and petD
50BIIa cleavages were similar in each case (data not shown). However, it took nearly twice
the time for HCSP41 to degrade 50% of the petD
50WT precursor (~8
min) than to degrade 50% of the petD
50BIIa precursor (~4 min,
Fig. 4B). Taken together, these data suggest that the
RNA-binding activity of CSP41 does not alter the specificity but
partially decreases the efficiency of its RNase activity, probably due
to conformational changes of bound CSP41, and/or a limitation of accessible substrate for free CSP41. The RNase activity clearly shows a
preference for stem-loop-containing RNAs over other single- and
double-stranded RNAs, which strongly suggests that some specific sequences or structures in the RNAs are efficiently recognized.
CSP41 Initially Cleaves the 3
Although HCSP41 cleaves petD50 RNA into
multiple shorter fragments, we noted in experiments such as the one
shown in Fig. 2A that certain products were much more
prevalent than others. To identify the initial cleavage products of
petD
50WT RNA, a time course of RNase digestion was carried out, as
shown in Fig. 5. The group of bands marked with
single asterisk in Fig. 5 comprises the earliest products,
appearing as early as the 0-min time point, with the cleavage
apparently occurring during the mixing of the reaction. Two other
groups of bands, marked with two or three asterisks in Fig. 5, were generated by HCSP41 within 2 min.
Subsequently, additional fragments were generated, either transiently
or stably. At the end of the 120-min reaction, all the larger products
were completely degraded and accumulated as very short RNA fragments seen near the bottom of the gel.
To determine the locations of the initial cleavage sites,
5-end-labeled petD
50WT RNA was synthesized and used as a substrate. A parallel assay used uniformly labeled petD
50WT RNA. Fig.
6A shows that by comparing the patterns
generated by the two substrates, it could be concluded that the group
of initial cleavage products of petD
50WT RNA marked with a
single asterisk in Fig. 5, and one of the groups of the
secondary cleavage products of petD
50WT RNA marked with two
asterisks, retained the original 5
-terminus. When calibrated
using an alkaline RNA ladder and a 47-nt RNA marker, the initial
cleavage sites were determined to be at nucleotides 133-137 and
141-142. The secondary cleavage sites were at nucleotides 114-115 and
119-120. Therefore, as shown in Fig. 6B, CSP41 initially cleaves petD
50 RNA within the 3
stem-loop and subsequently
upstream. Another group of secondary products, marked with three
asterisks in Fig. 5, and other shorter products, were not seen in
the assay with 5
-end-labeled substrate, indicating that they are
internal or 3
-end-containing products. Using the same approach, we
found that CSP41 initially cleaved 5
-end-labeled petD
50BIIa RNA at identical sites to those indicated in Fig. 6, demonstrating again that
the RNA-binding activity of CSP41 is not related to the specificity of
its RNase activity (data not shown).
The Ribonuclease Domain of CSP41 Is Located Between Amino Acids 73 and 191
Although it exhibits strong RNase activity, the primary
amino acid sequence of CSP41 does not show significant overall
similarity to any nuclease in the data bases. To begin locating domains
of CSP41 required for RNase activity, we constructed 3 deletion mutants of CSP41 and examined RNase activities of the resulting proteins. For this purpose, increasingly long C-terminal deletions of
CSP41 were generated by restriction enzyme digestion of
pQE30-csp41. Wild-type and mutant proteins (Fig.
7A) were expressed in E. coli, purified through an Ni-NTA column, and then refolded, but were not
subjected to further heparin-FPLC purification as in other experiments
described above, since the mutant proteins have very low affinity for
heparin. Their purities were examined in an SDS-polyacrylamide gel
(Fig. 7B), showing that there is only one major contaminant (marked with an asterisk) in each preparation. These protein
preparations were then tested for RNase activity in standard assays, as
determined by quantification of the remaining petD
50WT RNA over a
25-min time course. The results shown in Fig. 7C indicate
that HCSP41
S,
E, and wild-type HCSP41 quickly degrade petD
50
RNA, but much less RNase activity was displayed by HCSP41
P. A
comparison of their degradation profiles, shown in Fig.
8, reveals that both HCSP41
S and
E degrade
petD
50 RNA in the same manner as wild-type HCSP41 but that
HCSP41
P does not. Therefore, an essential domain for HCSP41 RNase
activity is located between amino acid residues 73 and 191. Based on
the results shown in Fig. 4B, the fact that HCSP41
S and
E degrade petD
50 RNA more quickly than wild-type HCSP41 may imply
that the C terminus of CSP41 is involved in the RNA-binding activity.
In this hypothesis, removal or partial removal of the C terminus would
eliminate or partially eliminate the RNA-binding activity of CSP41 and
thus enhance the efficiency of its RNase activity.
Although no overall similarity was noted between CSP41 and other
proteins, careful examination of the search results revealed a 50-amino
acid stretch from Glu127 to Trp176 of CSP41
which has 50% similarity to a sequence from Gln123 to
Trp172 of nuclease P1, a bacterial endonuclease.
Furthermore, this region contains the proposed active site of nuclease
P1 (Pro124Leu125His126), which can
be aligned with
Pro128Pro129His130 of CSP41
(Fig. 9A; 22). Because the 50-amino acid
sequence lies within the region containing the RNase domain of CSP41,
we decided to test whether this sequence contributed to CSP41
RNase function. We used the alanine-scanning approach (23) to make
mutant HCSP41A126, changing the wild-type sequence
Asp126Glu127Pro128Pro129His130
to
Ala126Ala127Pro128Pro129Ala130.
In this way, all of the hydrophilic residues in the immediate vicinity
of the putative active site were changed. HCSP41A126 was prepared in
the same way as the deletion mutant proteins. To avoid any variation in
the RNase activity due to expression and purification conditions,
especially to efficiency of refolding, a wild-type HCSP41 preparation
was concomitantly made as a control. The purities of the preparations
were examined in an SDS-polyacrylamide gel (Fig. 9B). In
contrast to our expectations, HCSP41A126 still exhibited strong
endoribonuclease activity (Fig. 9C), similar to that of
wild-type HCSP41. We conclude that RNase active site of CSP41 is not
contained within this sequence.
In this paper we have shown that CSP41, a spinach chloroplast
endoribonuclease, is able to cleave multiple single- and
double-stranded RNA molecules but not DNA. CSP41 appears to exhibit a
preference for stem-loop structures found at the 3 termini of
chloroplast mRNAs, and we have shown that for petD it
cleaves initially within the stem and subsequently proximal to it.
Since the stem-loop is known to stabilize chloroplast transcripts
(1-7), this cleavage could be a rate-determining step in
petD mRNA degradation.
3 to 5
exonucleolytic hydrolysis is a major pathway for mRNA
degradation in eukaryotes, prokaryotes, and organelles. Initially, an
endoribonuclease is usually required to overcome a 3
barrier, such as
a stem-loop structure or poly(A) tail, to generate free 3
ends
susceptible to exoribonucleases (11, 24, 25). RNase III and RNase E are
two of the best-characterized endoribonucleases in bacteria (9), and
eukaryotic homologues have been reported (26, 27). In bacteria, RNase
III and RNase E can cleave mRNAs within and upstream, respectively,
of mRNA 3
stem-loop structures (28-30). Since CSP41 is a
chloroplast endoribonuclease and cleaves initially within a stem-loop
structure, it is more similar to RNase III than to RNase E. However,
RNase III typically makes staggered, double-strand cleavages in
duplexed regions, whereas CSP41 cleaves only one strand in the duplex
region of petD
50 RNA. In addition, RNase III has a K+
optimum of 100-300 mM, conditions under which CSP41 RNase
activity is partially inhibited (Table I).
Several endoribonuclease activities, namely EndoC1, EndoC2, and a
putative RNase E-like protein, have been identified in spinach chloroplasts by our laboratory and others (15, 17). Although each
activity can cleave the 3-UTR of petD mRNA in
vitro, a comparison of cleavage sites and optional conditions for
their activities suggests that CSP41 is distinct from them. It is
important to note, however, that each of these other spinach
chloroplast activities was examined only in a complex mixture of
proteins. CSP41 also does not appear to be related to nuclease P1,
despite 50% similarity over a 50-amino acid region (Fig. 9).
Therefore, CSP41 could represent a new class or subclass of
ribonucleases.
Our data show that CSP41 degrades both single- and double-stranded RNAs
but prefers RNAs containing a stable stem-loop structure (Figs. 2 and
4). These data suggest that CSP41 could recognize certain special
nucleotide sequences or structural features of stem-loop regions. We
have noted that the two groups of initial cleavage sites are located
immediately upstream of two bulges in the petD stem-loop
structure (Fig. 6B). However, further characterization of
CSP41 RNase activity, especially using other stem-loop-containing RNAs
as substrates, is necessary to define its specificity. The 3 stem-loop
structure of chloroplast mRNA has been demonstrated to function as
a determinant of both correct 3
processing of pre-mRNA and
stabilization of mature mRNA by impeding the activities of 3
to 5
processive exonucleases (1-7). Our study suggests that CSP41 may be a
candidate to overcome the 3
secondary structure and initiate
degradation of upstream RNA sequences, possibly through the
polyadenylation-mediated pathway shown to function in bacteria (31, 32)
and chloroplasts (33, 34).
Although some RNases have RNA-binding activities to target their
substrates (35), the RNA-binding activity of CSP41 does not enhance
specificity or efficiency of its RNase activity in our in
vitro assay (Fig. 4), indicating that this RNA-binding activity
could be involved in a second function. CSP41 possesses two important
properties as an RNA-binding protein. First, it binds only to the
3-UTRs of certain chloroplast pre-mRNAs, such as those of
petD and rbcL, because it recognizes both the 3
stem-loop structure and a downstream AU-rich element (Fig. 3); and
second, it interacts with CSP55 and CSP29 to form a more stable complex containing the 3
-UTR of the selected pre-mRNA and multiple protein subunits (18, 19). Although the functions of CSP55 and CSP29 are still
unknown, it is possible that the function of CSP41 could be altered in
this larger complex by suppression of its RNase activity. In fact, our
data suggest that bound CSP41 may cleave substrate RNAs less
efficiently than free CSP41 (Fig. 4). Therefore, our data support one
of our previous (19) models that CSP41 could have two functions in
chloroplast mRNA metabolism. First, free CSP41 could directly
cleave chloroplast mRNA and pre-mRNA in the 3
stem-loop
structure to initiate bulk mRNA degradation, and second, as CSP41,
along with CSP55 and CSP29, forms a stable RNA-protein complex with the
3
stem-loop structure of a pre-mRNA, its RNase activity could be
suppressed and the entire complex could serve as a steric hindrance to
3
to 5
processive exoribonucleases, and thus ensure efficient 3
end
maturation.
We thank Rita-Ann Monde for the templates used to produce the psbA135 and rbcL115 transcripts. We acknowledge Dr. Gadi Schuster and members of the Stern laboratory for numerous helpful comments and suggestions.