|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 28, 25832-25838, July 11, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Secondary Structures Common to Chloroplast mRNA 3'-Untranslated Regions Direct Cleavage by CSP41, an Endoribonuclease Belonging to the Short Chain Dehydrogenase/Reductase Superfamily*
From the Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853
Received for publication, April 7, 2003 , and in revised form, May 5, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CSP41 (chloroplast stem-loop-binding protein of 41 kDa), a chloroplast endonuclease belonging to the SDR superfamily, preferentially cleaves stem-loop-containing RNAs in vitro. This potentially directs it to the 3'-ends of mature chloroplast mRNAs, which generally possess such structures. To understand the basis for this discrimination, the RNA elements directing CSP41 cleavage of petD RNA in vitro were dissected. Substrates containing fully base-paired stem-loops were optimal substrates, whereas deletion of part of the stem-loop decreased activity by 100-fold, and deletion of the distal arm of the stem-loop abolished cleavage, even in substrates containing the primary CSP41 cleavage site. Competition assays showed that the decrease in activity resulted from decreased affinity for the RNA by CSP41. Mutations of the residues at the scissile bond and mutations and deletions at the terminal loop of the stem had a moderate effect on activity but no effect on cleavage site specificity, suggesting that CSP41 has no sequence specificity. Titration of ethidium bromide into the assay decreased activity to a basal level of
18%, and introduction of a single base bulge into either arm of the
stem-loop decreased cleavage at the primary cleavage site by up to 70%. This
suggests that changing the structure of the helical stem has a mild effect on
activity. Deletion analysis of CSP41 suggests that the specificity domain lies
in the first 73 amino acids of the protein, a domain that also contains a
putative dehydrogenaselike mononucleotide binding motif. These results are
consistent with a broad role for CSP41 in the degradation of
stem-loop-containing mRNAs. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
In the chloroplasts of higher plants and green algae, gene expression is regulated primarily at the post-transcriptional level. Most chloroplast mRNAs contain an inverted repeat in their 3'-untranslated regions (UTRs)1 that can fold into a stable stem-loop structure. These stem-loops constitute highly specific recognition sites for nucleus-encoded RNA-binding proteins (1–3). In the absence of a stable stem-loop, or after their removal, transcripts become unstable (4), because the stem-loop and associated proteins impede the activity of processive 3'- to 5'-exonucleases (1, 5, 6). Therefore, removal of the stem-loop is likely to be a key regulatory step in chloroplast mRNA degradation (reviewed in Ref. 7). In light of this, a detailed understanding of how chloroplast ribonucleases distinguish their substrates is essential. However, although several chloroplast endoribonuclease activities (4, 8–10) and exoribonuclease activities (11) have been reported, only the exonuclease polynucleotide phosphorylase (12, 13) has undergone thorough analysis.
Previously, we reported the purification and characterization of CSP41 (chloroplast stem-loop-binding protein of 41 kDa), a ubiquitous endoribonuclease found in plant chloroplasts (14, 15). The amino acid sequence of CSP41 is >85% conserved in all plant species analyzed (data not shown), but it shares no homology with other proteins in the data bases. CSP41 also lacks known ribonuclease motifs but belongs to the short-chain dehydrogenase reductase (SDR) structural superfamily (16). This family consists of more than 1,600 proteins, including more than 130 in Arabidopsis thaliana (17). The best studied and closest relative of CSP41 in the SDR superfamily is Escherichia coli UDP-glucose epimerase. As such, CSP41 is predicted to contain a bidomain SDR Rossman fold. However, CSP41 lacks two sequence motifs required for epimerase activity (16, 18).
CSP41 was shown to cleave primarily within the stem-loop structures of several chloroplast RNA 3'-UTR substrates in vitro (15). Furthermore, whereas CSP41 was shown to cleave double-stranded RNA substrates, its activity was optimal with stem-loop-containing RNAs (15). Because stem-loop structures are known to be important for chloroplast mRNA stability, CSP41 was hypothesized to play a role in the initiation of RNA degradation. Three other members of the dehydrogenase family, glyceraldehyde-3-phosphate dehydrogenase and two dehydrogenases from the archaeon Solfolobus solfataricus, have been shown to have endoribonucleolytic activity. The most prominent cleavage sites of these enzymes were in loops and bulges of the predicted secondary structure of phage T7 R1.1 RNA (19), reminiscent of the prominent cleavage sites for CSP41 within the chloroplast petD 3'-UTR. Therefore, whereas the mechanistic details regarding substrate recognition and cleavage by SDR motif-containing proteins are still somewhat vague, the evidence suggests that this motif may represent a new type of ribonuclease domain capable of binding and cleaving double-stranded RNA substrates, particularly stem-loops. Here, we have attempted to determine the basis of this substrate preference, using CSP41 as a model.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Purification and Assay of CSP41—CSP41 was purified according to Yang and Stern (15). The enzyme was assayed at room temperature in 20 mM HEPES-KOH, pH 7.5, 10% glycerol, 20 fmol of petD RNA, and 20 mM MgCl2.
Preparation of Synthetic RNA Substrates—Templates encoding
the petD 3'-UTR RNA substrates used in this study were
contained between the SacI and EcoRV sites of pBluescript KS
(–). Synthetic RNAs were prepared according to Stern and Gruissem
(5) after linearizing templates
with HindIII. RNA substrates for CSP41 assays were synthesized with
2.5 µM [
-32P]UTP (3000 Ci/mmol) and 25
µM cold UTP. Trace-labeled RNAs were transcribed in the presence
of 8 nM [-32P]UTP and 0.5 mM cold UTP.
Substrates for footprinting were synthesized in the presence of 1
mM cold rNTPs and end-labeled with [
-32P]ATP
according to Yang and Stern
(15). Lengths of RNAs were as
follows: 
50, 208 nt; 18, 179 nt; 63, 168 nt; and
24, 152 nt. The DraI-linearized template produced a 92-nt
substrate.
Analysis of petD RNA Structure—105 cpm (roughly 20 fmol) of petD RNA was incubated in the presence of 20 mM HEPES-KOH, pH 7.5, 10% glycerol and 20 mM MgCl2 in a total volume of 10 µl. The RNA was then partially digested with either RNase A (2 ng), RNase T1 (1 unit), RNase T2 (0.24 unit), or RNase V1 (0.001 unit). The reactions were allowed to proceed for 1 min at room temperature and were then stopped with 50 µl of 5 mM aurintricarboxylic acid, 6 M urea, and 2% SDS. The reactions were extracted with phenol/chloroform and subsequently ethanol-precipitated at –20 °C in the presence of 20 µg of yeast tRNA. The precipitated RNA was collected by centrifugation and analyzed in a 6% denaturing polyacrylamide gel.
Inhibitor Titrations—The activity of CSP41 in the presence of variable concentrations of either ethidium bromide or actinomycin D was assayed as described above.
Inhibition Assays—CSP41 was assayed in the presence of 20
fmol of petD18 and unlabeled petD18, 63, or 24 at
1:1, 2:1, or 10:1 molar ratios (competitor/32P-petD18). The
resulting products were analyzed in 5% denaturing acrylamide gels. For
inhibition by tRNA, CSP41 was assayed in the presence of 20 fmol of
petD18 and the indicated molar quantities of unlabeled yeast tRNA. The
average molecular mass of the tRNA was assumed to be 2.5 x
104 g/mol.
Competition Assays with Inactive CSP41—A 10-µl sample of
CSP41 (1 mg/ml) was dialyzed against 100 mM MES-KOH, pH 6.5, and
10% glycerol. The equilibrated CSP41 was incubated in the presence of
1-ethyl-3-(3-dimethyl)-aminopropylcarbodiimide (EDAC) (100 mM) and
glycine methyl ester (25 mM) for 20 min at room temperature. The
reaction was stopped by dialysis against 20 mM HEPES-KOH, pH 7.5,
and 10% glycerol. The EDAC-modified CSP41 was used in the standard assay in
the presence of unmodified CSP41. Alternatively, CSP41P, a C-terminal
deletion mutant, was used as the protein competitor. This mutant has been
described previously (15).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
It was previously shown that whereas CSP41 cleaves efficiently within the double-stranded region of the petD RNA stem-loop or other stem-loop structures, an arbitrary double-stranded RNA was a poor substrate (15). The current studies were undertaken to gain insight into substrate recognition by this enzyme. We first used a series of deletion mutants of the preferred petD RNA substrate, as shown in Fig. 1A. petD
18 corresponds to the mature form of the
petD mRNA 3'-UTR, whereas petD63 and petD24
remove part or all of the stem-loop structure but still retain the primary
cleavage site for CSP41 at position 136. We assayed CSP41 with these
substrates to determine the minimal requirements for efficient and specific
cleavage at position 136 in the presence of 20 mM MgCl2.
We chose an enzyme/substrate ratio of 0.5 µg/20 fmol, within the range
where the rate response of CSP41 to substrate concentration is linear, and we
also determined that the accumulation of the 136-nt fragment was linear under
the conditions of our time course (data not shown). In subsequent experiments,
a single time point at 10 min was used to measure substrate cleavage.
Fig. 1B shows that
CSP41 cleaves petD18 efficiently into 136- and 43-nt products, whereas
the cleavage of petD63 at position 136 was only 1.5% the level seen for
petD18 (Fig.
1C). petD24 was cleaved at position 136 at a level
similar to that of petD63; however, cleavage occurred upstream of
position 136 (marked by an asterisk). A substrate that ends at the
petD translation termination codon (DraI) was not a
substrate for CSP41.
|
The stark difference in reactivity of 18 versus 63
could be due to sequence and/or structural differences. To map
single-versus double-stranded regions of these substrates,
5'-end-labeled petD18 and petD63 were digested with
sequence- and structure-specific endonucleases in the presence of 20
mM MgCl2, as shown in
Fig. 2. RNases A, T1, and T2
cleave in single-stranded regions only, whereas RNase V1 prefers helical RNA
substrates (20). The analysis
showed that upstream of the stop codon, the cleavage patterns were similar for
petD18 and petD63, suggesting that their secondary structures
are similar (data not shown). As predicted, there was a lack of petD18
cleavage with RNases A, T1, and T2 between positions 125 and 144 and between
positions 151 and 170, which correspond to the petD 3'-UTR
stems.
|
The cleavage pattern for petD63 differed from that of petD18
downstream of the stop codon. With petD63, RNase A and RNase T1 cleaved
in the proximal stem between positions 125 and 132, suggesting that
petD63 is single-stranded in this region. This and the lack of cleavage
at the G residues at positions 133–135 are consistent with the schematic
representation of petD63 in Fig.
2B, although minor cleavage by RNase T1 at position 151
may suggest flexibility in the structure. Since petD63 is largely
single-stranded at the base of its stem under the conditions of our
experiment, this suggests that the presence of a fully base-paired stem-loop
is required for optimal cleavage at position 136 by CSP41 and that the smaller
stem-loop still supports reduced but specific cleavage.
It was of interest to determine whether petD63 and petD24
were poor substrates because of inefficient binding by CSP41 or if binding was
unaffected but nonproductive in terms of promoting cleavage. In vivo,
nonproductive binding might trap CSP41 on nonsubstrate RNAs and in this sense
might be biologically unfavorable. To address the issue, competition
experiments were performed in which CSP41 was assayed in the presence of
radiolabeled petD18 and increasing concentrations of unlabeled
competitor substrates, as shown in Fig.
3. In Fig.
3A, the control reaction (lane –) shows
the cleavage of petD18 at position 136 in the absence of competitor
RNA. Using an equimolar amount of unlabeled petD18 RNA
(18–1X), the accumulation of the 136-nt product
decreased by 50% and decreased further as a function of increasing unlabeled
petD18. In the presence of a 10-fold molar excess of unlabeled
petD63, however, there was only a 15% decrease in accumulation of the
136-nt band compared with the control reaction (63–10X).
Only in the presence of a 100-fold molar excess of unlabeled petD63 was
the cleavage of petD18 significantly reduced (shown in
Fig. 7C). There was no
detectable decrease in cleavage of petD18 in the presence of a 10-fold
molar excess of petD24 (24–10X). The results of
the competition assays are summarized in
Fig. 3B, which was
repeated with unlabeled yeast tRNA. 50% inhibition of petD18 cleavage
by CSP41 occurred only in the presence of a 400-fold molar excess of tRNA.
Taken together, these data suggest that a fully basepaired stem-loop is
required for a high affinity interaction between CSP41 and petD RNA
and that stem-loops in yeast tRNA do not possess the optimal structure
required for binding by CSP41.
|
),
petD
), or petD
) was normalized
accordingly. The curves show the trend in the data and do not
represent a least-squares fit to the data.
|
To further assess the importance of the double-stranded RNA stem-loop in
recognition and cleavage of petD by CSP41, cleavage was measured in
the presence of increasing concentrations of ethidium bromide, which
intercalates into both dsRNA and double-stranded DNA. Intercalation of EtBr
would be expected to distort the helix of 18, changing the spatial
relationship of nucleotides potentially recognized by CSP41. If this treatment
impeded cleavage, it might be concluded that CSP41 recognized specific
sequences and/or required an undistorted helix for cleavage.
As shown in Fig. 4A, increasing EtBr concentrations caused corresponding decreases in cleavage at position 136. (Several secondary CSP41-catalyzed cleavages are also visible in this gel; however, they were not reproducible (marked with asterisks).) To control for inhibition due to direct binding and inactivation of CSP41 by ethidium, the experiment was repeated in the presence of increasing concentrations of actinomycin D. Actinomycin D, like ethidium, is a hydrophobic DNA intercalator but is not an RNA intercalator. Propidium, another hydrophobic intercalating molecule similar in structure to ethidium, has been shown to inactivate RNase A nonspecifically via interactions at an apolar site on the enzyme, whereas RNase III was not inactivated by actinomycin D under similar conditions (21, 22). When CSP41 was assayed with increasing levels of actinomycin D, cleavage at position 136 was unaffected (Fig. 4B). Furthermore, the same results were obtained whether the reaction was started with the addition of CSP41 or with RNA, suggesting that preincubation of the enzyme with EtBr did not directly inactivate CSP41.
|
The percentage of cleavage of petD RNA at position 136 was plotted
as a function of EtBr concentration (Fig.
4C). The cleavage decreases as a function of
concentration and plateaus at a level of 18%. The concentration giving
half-maximal inhibition of cleavage was 3 µM. This is
similar to the dissociation constant of 2.4 µM for EtBr binding
to tRNA (23). Taken together,
these data suggest that EtBr intercalates into the double-stranded stem of the
petD 3'-UTR and disrupts cleavage, probably by changing the
structure of the double-stranded stem.
As an independent test of the importance of three-dimensional stem
structure, single base bulges were introduced independently into each strand
of the inverted repeat. The petD19 RNA contains an A insertion at position
166, and petD20 contains a U insertion at position 130. The structures tested
are shown in Fig. 5A,
and the results of cleavage assays are shown in
Fig. 5B. The cleavage
position of these substrates remained the same; however, compared with the
control experiment with petD18, cleavage of petD19 and petD20 was
reduced by 30 and 70%, respectively. This is consistent with the results
obtained with EtBr, where cleavage at position 136 was significantly reduced
but not abolished. This further emphasizes that the formation of a fully
base-paired stem-loop enhances cleavage at position 136.
|
To gain additional insight into the relationship between RNA
sequence/structure and cleavage efficiency/specificity, the cleavage rate and
sequence specificity of CSP41 was measured with the mutant substrates shown in
Fig. 6A. Because these
mutations were introduced into petD50, the accumulation of the 136-nt
product of each mutant was normalized to this substrate
(Figs. 6, B and
C). In petD2, the C residues at positions 160 and 161
have been mutated to G residues, which causes a complete unwinding of the
minor stem loop between positions 141 and 154
(4), whereas the primary CSP41
cleavage site is retained. In petD10, the A residues at positions 136 and 137
that flank the CSP41-targeted scissile bond have been mutated to U. petDd5 and
petDd6 contain internal deletions in the proximal stem downstream of the
scissile bond. CSP41 cleaved petD2, petD10, petDd5, and petDd6 with the same
specificity as petD50 (data not shown). As shown in
Fig. 6C, the cleavage
at position 136 was 30% higher and 50% higher with petDd5 and petDd6,
respectively, than with 50 (wild type). This suggests that mutations
downstream of the scissile bond have a moderate effect on the efficiency of
cleavage at position 136 and that neither the sequence nor the structure above
the scissile bond, nor the nucleotides at positions 136 and 137, direct
specific cleavage at position 136. Overall, the experiments to this point
paint a picture of an enzyme with significant structural but little sequence
specificity.
|
).
Numbering is referred to under "Results" and begins at the
5'-end of the in vitro transcript. B, cleavage of
petD
Although CSP41 is a member of the SDR structural family based on its
sequence, this does not allow the a priori conclusion that SDR
domains are involved in its function. To determine whether its SDR domains are
important for specificity in binding and cleavage at position 136, we
performed competition experiments in which wild type (full-length) CSP41 was
assayed in competition with either inactivated full-length CSP41 or a CSP41
deletion mutant. We first performed a control experiment in which the
competitor was an inactive form of CSP41. Wild type CSP41 was modified with
EDAC and glycine methyl ester, which functions by modifying acidic (Asp/Glu)
residues and inactivates the enzyme (data not shown). As shown in
Fig. 7A, increasing
amounts of unmodified CSP41 cause a corresponding increase in degradation of
the full-length petD18 substrate as expected, whereas adding
EDAC-modified CSP41 to the standard assay resulted in a decrease in cleavage.
This suggests that catalytically inactivated CSP41 has the same binding
specificity as the unmodified enzyme and that it forms a specific complex with
petD18 that prevents cleavage by unmodified enzyme. We cannot
completely rule out that EDAC-modified CSP41 inactivates the unmodified
enzyme, for example by dimerizing, although unmodified CSP41 acts as a monomer
when monitored by native gel electrophoresis or gel filtration (data not
shown).
An interesting observation in this experiment is that at increasing
concentrations of CSP41, there is little additional accumulation of the 136-nt
fragment, which would be expected given the increase in cleavage rate of the
full-length RNA. It is possible that further cleavage of the 136-nt RNA could
occur at these high enzyme concentrations. When dilutions of CSP41 are made
below 0.5 µg/reaction, the amount of CSP41 added to the standard assay,
both the degradation of full-length substrate and the accumulation of the
136-nt fragment decrease in a linear fashion as a function of decreasing CSP41
(data not shown). Furthermore, at 0.5 µg of CSP41, petD24, which is
nearly identical to the 136-nt product, was cleaved at 1% the rate of
petD18 but at a position several nucleotides upstream of position 136.
The 136-nt product may be degraded in a similar manner at high CSP41
concentrations.
The experiment was repeated with increasing amounts of CSP41P, a
deletion mutant that contains only the N-terminal 73 amino acids
(15). This portion of CSP41
contains three highly conserved motifs, including the 
mononucleotide motif responsible for binding NAD(P)H in dehydrogenases
(16). At least part of the
CSP41 active site is deleted, because CSP41P is not catalytically
active (15).
Fig. 7B shows that
CSP41P inhibits cleavage of petD18 at position 136 by wild type
(full-length) CSP41. 50% inhibition of CSP41 activity occurred at a 1:16 molar
ratio of CSP41P to wild type CSP41, suggesting that CSP41P has a
16-fold higher affinity for petD18. The reason for this increased
affinity is unknown, but it may be related to the lack of a complete catalytic
site. These data also suggest that the specificity determinant of CSP41 may
lie in the N-terminal 73 amino acids.
The effect of added CSP41P shown in
Fig. 7B could result
from specific or nonspecific binding of CSP41P to the RNA. To test the
specificity of CSP41P binding, we repeated the assay in the presence of
different RNA competitors. It was expected that if CSP41P bound the
petD stem-loop specifically, competition with unlabeled petD63
would not alleviate inhibition of (wild type) CSP41 cleavage by CSP41P,
since 63 is not a substrate for specific cleavage
(Fig. 1). On the other hand, if
the binding of CSP41P were nonspecific, then unlabeled petD63
would compete with petD18 for binding of CSP41P and alleviate
inhibition of wild type CSP41 by CSP41P. In the presence of molar
equivalents of wild type CSP41 and CSP41P, cleavage at position 136 of
petD18 is almost completely abolished compared with the control
reaction (Fig. 7B,
third lane from left). Furthermore, in the presence of a 100-fold
molar excess of unlabeled petD63 over radiolabeled petD18,
cleavage at position 136 of petD18 was similarly affected
(Fig. 7C). In the
presence of both an 8-fold molar excess of wild type CSP41 over CSP41P
and a 100-fold molar excess of unlabeled petD63 over petD18,
there was no cleavage at position 136. This suggests that CSP41P has
the same binding specificity as wild type CSP41 and that affinity is highest
for a fully base-paired stem-loop.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Previous results suggested that whereas CSP41 could recognize arbitrary dsRNA substrates, its cleavage activity and specificity were much lower than in the presence of petD RNA. This suggested that CSP41 recognized specialized structures and/or sequences within the petD RNA (15). Here, we have expanded on its substrate specificity and determined the RNA structural elements necessary for recognition and cleavage within the stem-loop.
A key question was whether the sequence at the scissile bond was important for cleavage, since such sequence contexts might be relatively rare in the chloroplast. In petD10, the adenine residues at positions 136 and 137, the primary cleavage sites for CSP41, were mutated to uracil (Fig. 6). This had the double effect of changing the identity of the bases at these positions and changing them from purines to pyrimidines. The cleavage of petD10 was the same as for wild type petD RNA, suggesting that CSP41 has no primary sequence specificity or preference for a particular type of residue. This is different from general endoribonucleases such as RNase A and RNase T1, which have a preference for cleavage after purines and after guanosine residues, respectively, or RNase T2, which has a slight preference for adenine residues but will cleave after any residue (24).
Results of cleavage assays with the substrates petD24 and
petD63 (Fig. 1) suggest
that there is no consensus sequence upstream of the petD stem-loop
that can alone direct binding and cleavage. The results of cleavage assays
containing one or multiple point mutations in several positions within the
stem-loop further suggest that there is no consensus sequence within the stem
that is recognized by CSP41 (Fig.
6). One implication of these results is that in vivo,
CSP41 might target many 3'-UTRs, since most chloroplast mRNAs terminate
in stem-loops. This would further implicate CSP41 broadly in chloroplast mRNA
decay rather than as a petD-specific or highly specialized
enzyme.
Although we did not identify specific RNA residues that directed CSP41
cleavage, the deletion mutants showed the importance of a double-stranded
stem. The optimal petD substrate was petD18, which represents
the mature petD RNA in vivo. Cleavage at position 136 of
petD63, which retains three base pairs below the scissile bond, was
100-fold lower, although specificity for position 136 was retained. With
petD24, lacking any stem base pairs, cleavage at position 136 was
abolished. This is similar to the structural requirements of the substrates of
yeast Rnt1p and E. coli RNase III, both of which require a
double-stranded RNA region for recognition and cleavage. Furthermore, Rnt1p
cleavage has been shown to require a duplex region on either side of the
cleavage site, whereas the efficiency of cleavage is enhanced by a complete
stem below the cleavage site
(25). The results of
competition experiments suggested that the low cleavage activity with
petD63 and with petD24 reflects the inability of CSP41 to bind
the mutated petD RNA rather than nonproductive binding
(Fig. 3). This suggests that
CSP41 contains a motif capable of discriminating between single- and
double-stranded RNA substrates. To our knowledge, this is the first
description of such a binding activity in an SDR protein.
The importance of the helical nature of the stem-loop was tested using either an intercalating dye, EtBr (Fig. 4), or by introducing single base bulges that could alter the helical structure of the stem (Fig. 5). Cleavage decreased in the presence of EtBr, which has been shown to cause unwinding, lengthening, and local distortion of RNA double helices (22). The fact that CSP41 cleavage was not completely abolished by saturating ethidium bromide shows that it was not inhibiting cleavage by simply intercalating at the scissile bond and preventing cleavage, a phenomenon that has been observed in similar experiments with E. coli RNase III (22).
We complemented these data by introducing single base bulges into the stem midway between the base of the stem and the primary CSP41 cleavage site, which caused modest decreases in cleavage at position 136 (Fig. 5). This suggests that whereas CSP41 requires a double-stranded stem for recognition and cleavage at a particular site, the absolute requirement for an A-form helix is relaxed with CSP41, and the RNA recognition motif on the enzyme can recognize several forms of helical RNA. This is also true for the double-stranded RNA binding motif, commonly found in dsRNA-binding proteins and double-strand-specific ribonucleases. For example, both human interferon-induced dsRNA-induced protein kinase and Xenopus RNA-binding protein A were found to bind to RNAs with secondary structure defects, provided the helix had an overall A-form geometry (26, 27). The relatively loose requirement for A-form RNA is reflected in the fact that CSP41 is able to cleave petD, psbA, and rbcL RNA stem-loops at similar rates (15). These substrates neither share primary sequence nor are predicted to share a common tertiary structure. However, unlike the dsRNA-specific (and double-stranded RNA binding motifcontaining) endoribonuclease Rnt1p from yeast (28) or Staufen from Drosophila (29), CSP41 does not appear to require specific interactions with the terminal loop on the stem-loop, because mutations that either alter the three-dimensional structure of the loop or modify the sequence of the terminal loop caused at most a 50% change in the level of cleavage of petD at position 136 (Fig. 6). With Rnt1p, for example, mutation of the AGGA tetraloop terminating the 25 S rRNA 3'-ETS substrate to GUGA causes at least a 4-fold decrease in both affinity and cleavage rate (30).
CSP41 is predicted to be a member of the short chain
dehydrogenase/reductase superfamily
(16). Comparisons of the
sequences and three-dimensional structures of many proteins in this family
show that they are structurally related despite significant divergence in
their amino acid sequences. We have shown here that the N-terminal 73 amino
acids of CSP41 contain a domain with a high affinity and specificity for the
petD double-stranded stem-loop
(Fig. 7). Multiple
expectation-maximum for motif elicitation
(MEME) analysis using a training set of 195 SDR proteins suggested that three
highly conserved structural motifs lie in this domain of CSP41. MEME is an
artificial intelligence-based motif analysis tool that identifies the
conserved regions that are characteristic of the data set, given a set of
unaligned sequences (16).
Further analysis showed that one of the conserved motifs overlapped with the
mononucleotide binding fold in dehydrogenases
(16).
Several dehydrogenases have previously been shown to be sequence-specific
RNA-binding proteins. Glyceraldehyde-3-phosphate dehydrogenase was also shown
to have a high affinity for tRNA
(31,
32). Recently, it was reported
that yeast glyceraldehyde-3-phosphate dehydrogenase and two dehydrogenases
from the archaeon S. solfataricus, Acd-1 and Acd-5, are
endoribonucleases (19). In the
S. solfataricus enzymes, the active site of the enzyme was localized
to the first mononucleotide binding motif, contained within the first 70 amino
acids of the enzyme. However, direct comparisons between these dehydrogenases
and CSP41 must be made with caution. The first mononucleotide binding motif of
CSP41, which lies within the sequence of CSP41P, does not have
catalytic activity, since activity requires the first 191 amino acids of the
protein. Furthermore, CSP41 activity absolutely requires
Mg2+, whereas glyceraldehyde-3-phosphate dehydrogenase,
Acd-1, and Acd-5 do not (19).
Neither the endonucleolytic activity nor the cleavage specificity of CSP41 is
inhibited by mono- or
dinucleotides.2 These
data suggest that, whereas CSP41 contains a putative mononucleotide binding
motif, the affinity of this motif for nucleotides is significantly reduced in
CSP41.
|
FOOTNOTES |
|---|
* This work was supported by Department of Energy Biosciences Program Award DE-FG02-90ER20015. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Boyce Thompson Institute for Plant
Research, Cornell University, Tower Rd., Ithaca, NY 14853. Tel.: 607-254-1306;
Fax: 607-255-6695; E-mail:
ds28{at}cornell.edu.
1 The abbreviations used are: UTR, untranslated region; dsRNA,
double-stranded RNA; EDAC, 1-ethyl-3-(3-dimethyl)aminopropylcarbodiimide; nt,
nucleotide; SDR, short-chain dehydrogenase/reductase; MES,
4-morpholineethanesulfonic acid.
2 T. J. Bollenbach and D. B. Stern, unpublished data.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
- Stern, D. B., Jones, H., and Gruissem, W. (1989)
J. Biol. Chem. 264,
18742–18750
[Abstract/Free Full Text] - Chen, H. C., and Stern, D. B. (1991) Mol.
Cell. Biol. 11,
4380–4388
[Abstract/Free Full Text] - Memon, A. R., Meng, B., and Mullet, J. E. (1996) Plant Mol. Biol. 30, 1195–1205[CrossRef][Medline] [Order article via Infotrieve]
- Chen, H., and Stern, D. B. (1991) J. Biol.
Chem. 266,
24205–24211
[Abstract/Free Full Text] - Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145–1157[CrossRef][Medline] [Order article via Infotrieve]
- Stern, D. B., and Gruissem, W. (1989) Plant Mol. Biol. 13, 615–625[CrossRef][Medline] [Order article via Infotrieve]
- Monde, R. A., Schuster, G., and Stern, D. B. (2000) Biochimie (Paris) 82, 573–582[Medline] [Order article via Infotrieve]
- Klaff, P. (1995) Nucleic Acids
Res. 23,
4885–4892
[Abstract/Free Full Text] - Kudla, J., Hayes, R., and Gruissem, W. (1996) EMBO J. 15, 7137–7146[Medline] [Order article via Infotrieve]
- Nickelsen, J., and Link, G. (1993) Plant J. 3, 537–544[CrossRef][Medline] [Order article via Infotrieve]
- Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W. (1996) EMBO J. 15, 1132–1141[Medline] [Order article via Infotrieve]
- Yehudai-Resheff, S., Hirsh, M., and Schuster, G.
(2001) Mol. Cell. Biol.
21,
5408–5416
[Abstract/Free Full Text] - Baginsky, S., Shteiman-Kotler, A., Liveanu, V., Yehudai-Resheff, S., Bellaoui, M., Settlage, R. E., Shabanowitz, J., Hunt, D. F., Schuster, G., and Gruissem, W. (2001) RNA (N. Y.) 7, 1464–1475[Abstract]
- Yang, J., Schuster, G., and Stern, D. B. (1996) Plant Cell 8, 1409–1420[Abstract]
- Yang, J., and Stern, D. B. (1997) J. Biol. Chem. 272, 12784–12880
- Baker, M. E., Grundy, W. N., and Elkan, C. P. (1998) Biochem. Biophys. Res. Commun. 248, 250–254[CrossRef][Medline] [Order article via Infotrieve]
- Kallberg, Y., Oppermann, U., Jornvall, H., and Persson, B. (2002) Protein Sci. 11, 636–641[CrossRef][Medline] [Order article via Infotrieve]
- Bellamacina, C. R. (1996) FASEB J. 10, 1257–1269[Abstract]
- Evguenieva-Hackenberg, E., Schiltz, E., and Klug, G.
(2002) J. Biol. Chem.
277,
46145–46150
[Abstract/Free Full Text] - Lowman, H. B., and Draper, D. E. (1986) J.
Biol. Chem. 261,
5396–5403
[Abstract/Free Full Text] - McGrath, M., Cascio, D., Williams, R., Johnson, D., Greene, M., and McPherson, A. (1987) Mol. Pharmacol. 32, 600–605[Abstract]
- Calin-Jageman, I., Amarasinghe, A. K., and Nicholson, A. W.
(2001) Nucleic Acids Res.
29,
1915–1925
[Abstract/Free Full Text] - Torgerson, P. M., Drickamer, H. G., and Weber, G. (1980) Biochemistry 19, 3957–3960[CrossRef][Medline] [Order article via Infotrieve]
- Uchida, T., and Egami, F. (1967) J.
Biochem. (Tokyo) 61,
44–53
[Abstract/Free Full Text] - Abou Elela, S., and Ares, M., Jr. (1998) EMBO J. 17, 3738–3746[CrossRef][Medline] [Order article via Infotrieve]
- Bevilacqua, P. C., George, C. X., Samuel, C. E., and Cech, T. R. (1998) Biochemistry 37, 6303–6316[CrossRef][Medline] [Order article via Infotrieve]
- Ryter, J. M., and Schultz, S. C. (1998) EMBO J. 17, 7505–7513[CrossRef][Medline] [Order article via Infotrieve]
- Lebars, I., Lamontagne, B., Yoshizawa, S., Aboul-Elela, S., and Fourmy, D. (2001) EMBO J. 20, 7250–7258[CrossRef][Medline] [Order article via Infotrieve]
- Ramos, A., Grunert, S., Adams, J., Micklem, D. R., Proctor, M. R., Freund, S., Bycroft, M., St. Johnston, D., and Varani, G. (2000) EMBO J. 19, 997–1009[CrossRef][Medline] [Order article via Infotrieve]
- Nagel, R., and Ares, M., Jr. (2000) RNA (N. Y.) 6, 1142–1156[Abstract]
- Singh, R., and Green, M. R. (1993)
Science 259,
365–368
[Abstract/Free Full Text] - Nagy, E., and Rigby, W. F. C. (1995) J.
Biol. Chem. 270,
2755–2763
[Abstract/Free Full Text]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. J. Ytterberg, J.-B. Peltier, and K. J. van Wijk Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes Plant Physiology, March 1, 2006; 140(3): 984 - 997. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nishimura, E. A. Kikis, S. L. Zimmer, Y. Komine, and D. B. Stern Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts PLANT CELL, November 1, 2004; 16(11): 2849 - 2869. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |