Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037
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Abstract |
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The 5' untranslated region of the chloroplast psbA mRNA, encoding the D1 protein, is processed in Chlamydomonas reinhardtii. Processing occurs just upstream of a consensus Shine-Dalgarno sequence and results in the removal of 54 nucleotides from the 5' terminus, including a stem-loop element identified previously as an important structure for D1 expression. Examination of this processing event in C. reinhardtii strains containing mutations within the chloroplast or nuclear genomes that block psbA translation reveals a correlation between processing and ribosome association. Mutations within the 5' untranslated region of the psbA mRNA that disrupt the Shine-Dalgarno sequence, acting as a ribosome binding site, preclude translation and prevent mRNA processing. Similarly, nuclear mutations that specifically affect synthesis of the D1 protein specifically affect processing of the psbA mRNA. In vitro, loss of the stem-loop element does not prohibit the binding of a message-specific protein complex required for translational activation of psbA upon illumination. These results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.
Key words: translation; mRNA processing; chloroplast; ribosome binding sequence; psbA ![]() |
Introduction |
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PHOTOSYSTEM II is comprised of proteins encoded
within both the nuclear and chloroplast genomes.
The expression of these proteins is regulated in response to light at the level of transcription, RNA processing, message stability, translation, and protein turnover (reviewed in Gillham et al., 1994; Mayfield et al., 1995
;
Rochaix, 1996
). For many chloroplast-encoded proteins,
translation appears to be the rate-limiting step of gene expression. Chloroplast mRNA levels remain relatively unchanged throughout dark/light transitions whereas protein
synthesis rates increase dramatically upon illumination (Fromm et al., 1985
; Deng and Gruissem, 1988
; Malnoë
et al., 1988
). Genetic and biochemical evidence suggests
that nuclear-encoded factors mediate this light-regulated
translation of chloroplast-encoded proteins (Jensen et al.,
1986
; Kuchka et al., 1988
; Rochaix et al., 1989
; Girard-Bascou et al., 1992
; Wu and Kuchka, 1995
; Yohn et al., 1996
;
Zerges et al., 1997
; Yohn et al., 1998b
). These nuclear factors typically interact with specific elements within the 5'
untranslated regions (UTRs)1 of the chloroplast mRNAs
(Rochaix et al., 1989
; Sakamoto et al., 1994a
; Zerges and
Rochaix, 1994
; Stampacchia et al., 1997
; Zerges et al.,
1997
).
The D1 protein, a core component of Photosystem II, is
encoded by the chloroplast psbA gene. Throughout the
light phase of photosynthesis, the D1 protein is subject to
photodamage and is rapidly turned over (reviewed in Barber and Andersson, 1992). D1 inactivation is compensated
by a 50-100-fold increase in the rate of D1 protein synthesis in response to light without a corresponding increase in
psbA mRNA levels (Fromm et al., 1985
; Klein et al., 1988
;
Malnoë et al., 1988
; Krupinska and Apel, 1989
). The use of
reporter gene constructs in tobacco has demonstrated that
the psbA 5' UTR is sufficient to confer light-dependent
translational regulation in vivo (Staub and Maliga, 1994
).
An in vitro translation system derived from tobacco chloroplasts has identified critical regulatory elements for D1
synthesis in the psbA 5' UTR including potential ribosome
binding sequences (RBS), an AU-box, and to a lesser extent, an upstream stem-loop element (Hirose and Sugiura, 1996
). A stem-loop element has also been mapped within
the 5' UTR of the spinach psbA mRNA (Klaff and Gruissem, 1995
; Klaff et al., 1997
). This element encompasses a
putative RBS, an endonucleolytic cleavage site for mRNA
decay (Klaff, 1995
), and sequences recognized by stromal
proteins (Klaff et al., 1997
; Alexander et al., 1998
).
In the unicellular green algae Chlamydomonas reinhardtii, similar elements within the 5' UTR have been
identified as important for psbA expression. Among these
elements is a stem-loop structure immediately upstream of
a consensus Shine-Dalgarno (SD) sequence. Mutational
analysis of the stem-loop region has shown that this element serves a role in psbA expression, although the exact
nature of this role remains unresolved (Mayfield et al.,
1994). Deletion of the SD sequence prevents ribosome association with the psbA mRNA and synthesis of the D1
protein, consistent with its proposed function as an RBS
(Mayfield et al., 1994
).
A complex of proteins, thought to serve as light-dependent translational activators, specifically recognizes the C.
reinhardtii psbA 5' UTR (Danon and Mayfield, 1991). The
binding activity of this complex is modulated in response
to changes in photosynthetic activity via a redox switch
(Danon and Mayfield, 1994
; Kim and Mayfield, 1998
). The
principal RNA-binding (RB) protein in this complex is a
chloroplast-localized poly(A)-binding protein (cPABP) homologue (Yohn et al., 1998a
). Several nuclear mutants
have been isolated in which the loss of the cPABP is accompanied by the absence of D1 protein synthesis due to a
block in psbA mRNA association with polyribosomes
(Yohn et al., 1996
; Yohn et al., 1998b
). A previous study
suggested that this complex binds the stem-loop element
upstream of the SD sequence (Danon and Mayfield, 1991
). A model has been proposed (summarized in Mayfield et
al., 1995
) in which the stem-loop element acts as a translational attenuator in the dark, preventing ribosome association with the SD sequence. Upon illumination, and subsequent increase in photosynthetic activity, binding activity
of the protein complex increases and stimulates translation initiation by disrupting the repressive stem-loop element or by providing a platform for ribosome association.
However, examination of psbA transcripts from C. reinhardtii indicated that in vivo the majority of this message
lacks sequences upstream of the SD sequence including
the stem-loop element (Erickson et al., 1984; Nickelsen
et al., 1994
; Shapira et al., 1997
). In this study, we investigate the differential accumulation of psbA mRNAs containing different 5' termini. These different 5' UTRs likely
result from the processing of the 90-nucleotide (nt) 5' UTR to generate a new 5' terminus 36 nt upstream of the
initiation codon. Processing of the psbA 5' UTR is shown
to be closely correlated with ribosome association. In the
absence of a competent and accessible SD sequence, D1
protein is not synthesized and the psbA 5' UTR is not processed. Nuclear mutations that block D1 translation, in
conjunction with reduced association of psbA mRNA with
ribosomes, also reduce psbA processing. However, removal of the stem-loop element as a consequence of processing does not prohibit the binding of the nuclear-encoded
protein complex to this psbA 5' UTR in vitro, nor does it
preclude dynamic light-dependent translational regulation
mediated by the psbA RB complex. Based on these observations, we propose a model for psbA mRNA maturation
in which 5' end formation does not serve as a prerequisite
for initiation complex formation but rather processing of
the 5' UTR occurs in conjunction with the early stages of
ribosome assembly at the RBS.
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Materials and Methods |
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Cell Growth Conditions
Unless otherwise noted, all C. reinhardtii strains were grown at 25°C under constant light in complete media (Tris-acetate-phosphate; Harris,
1989) to a density of ~106 cells/ml. Cells were harvested by centrifugation
at 4°C for 5 min at 4,000 g, washed with H2O, and pelleted again at 4,000 g for 5 min at 4°C. Cell pellets were frozen in liquid N2 and stored at
70°C.
RNA Isolation
Total and polyribosome-associated RNAs were prepared according to
published protocols (Barkan, 1988; Cohen et al., 1998
). To prepare membrane-associated RNA, the ground frozen cell pellet was twice extracted
with extraction buffer lacking the detergents Triton X-100 and polyoxyethelene-10-tridecyl-ether to remove soluble RNA. The insoluble pellet
was then resuspended in extraction buffer containing these detergents and
used to prepare polyribosome-associated RNA.
Primer Extension Analysis
Approximately 1 pmol of 5'-radiolabeled DNA oligonucleotide complementary to positions +47 to +32 of the psbA coding region (5'-CGAGCCCATAGGCTAG-3') or positions +66 to +49 of the psbD coding region (5'-AAGCCAGTCATCAGCGTC-3') was annealed to 5-25 µg RNA in a 7-µl mixture containing 50 mM Tris (pH 8.3), 60 mM NaCl, and 10 mM DTT by slow cooling from 80°C to room temperature. The reaction mixture was incubated at 42°C for 30 min after addition of 8 µl reaction mixture (50 mM Tris, pH 8.3, 60 mM NaCl, 10 mM DTT, 15 mM MgCl2, 1.25 mM each dNTP, and 0.2 U AMV reverse transcriptase; Life Sciences Inc., St. Petersburg, FL). The RNA was degraded by addition of 20 µl degradation buffer (50 mM Tris, pH 7.5, 0.1% SDS, and 7.5 mM EDTA, pH 8.0) and 3.5 µl 3 M KOH followed by incubation at 90°C for 3 min then 42°C for 25 min. The sample was neutralized upon addition of 6 µl acetic acid and 10 µl 3 M NaOAc (pH 5.3), EtOH precipitated, and resuspended in gel loading buffer. Products were separated by electrophoresis in a 7.5% polyacrylamide/8 M urea gel and quantified by PhosphorImager analysis. Samples were loaded to achieve approximately equal signals and do not reflect the relative amounts of psbA mRNA in each strain of C. reinhardtii.
Analysis of psbA 5' UTR Length by Northern Analysis after RNase H Cleavage
100 pmol of a DNA oligonucleotide complementary to positions +171 to
+156 of the psbA coding region (5'-TGGCGGAGCAGCGATG-3') was
annealed to 5 µg wild-type total RNA or 15 µg hf261 total RNA in a 3.5-µl
mixture containing 0.5 µl buffer (200 mM Tris, pH 7.5, 200 mM KCl, 1 mM EDTA, pH 8.0, and 1 mM DTT) and 0.1 U PRIME RNase inhibitor
(5 Prime 3 Prime, Inc., Boulder, CO) by slow cooling from 80°C to
room temperature. RNA cleavage was initiated upon addition of 0.5 µl
100 mM MgCl2, 0.5 µl 0.2 U/µl PRIME RNase inhibitor, and 0.5 µl RNase H
(2.2 U/µl; GIBCO BRL, Gaithersburg, MD). Reactions were incubated at
37°C for 30 min. Products were separated by electrophoresis in a 5% polyacrylamide/8 M urea gel. RNA blotting and hybridization with a random-primed, [32P]-labeled psbA 5' cDNA fragment (HindIII-ClaI) were performed as described previously (Mayfield et al., 1994
).
Gel Mobility Shift Assay
Approximately 1 µg total protein from C. reinhardtii, partially purified by
heparin-agarose chromatography according to Cohen et al. (1998), was incubated for 10 min at room temperature with 0.5 U PRIME RNase inhibitor in a total volume of 8 µl dialysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM KOAc, 0.2 mM EDTA, pH 8.0, 2 mM DTT, 20% glycerol) supplemented with 4 mM MgCl2. To each sample, 0.08 pmol of in vitro transcribed [32P]-labeled RNA (beginning with two G residues added 90 bases
[
90] or 36 bases [
36] upstream of the start codon and ending 171 bases downstream of the start codon), 20 µg of wheat germ tRNA (Sigma), and
3 µg of FuD7 (a C. reinhardtii strain lacking the psbA gene) total RNA
were added and the reaction was incubated at room temperature for 10 min. Where indicated, 8 pmol unlabeled in vitro transcribed RNA was
also added as a competitor. RNA-protein complexes were separated in a
5% nondenaturing polyacrylamide gel.
UV Cross-linking Assays
UV cross-linking assays were performed as gel mobility shift assays with
the following alterations. Approximately 5 µg heparin-agarose purified
protein was incubated with 1.0 pmol of internally labeled RNA (90 or
36) generated by in vitro transcription using both [
-32P]ATP and
[
-32P]UTP. After complex formation, the reactions were irradiated with
short-wave UV light for 1 h at 4°C. The RNA was digested at 55°C for 45 min after addition of urea, EDTA (pH 8), and RNase A to final concentrations of 3 M, 3.75 mM, and 0.5 mg/ml, respectively. Labeled proteins
were separated by SDS-PAGE and visualized using a PhosphorImager.
RNA Affinity Chromatography (RAC) and Immunoblot Analysis
RAC was performed as described by Cohen et al. (1998). In brief, 125 µg
of in vitro transcribed RNA (
90 or
36) was coupled to 100 µl amino gel
1702 (Sterogene Bioseparations Inc., Carlsbad, CA). 1 ml heparin-agarose
purified protein was passed over the resulting columns, washed, and recovered by elution with a high salt buffer. Equal quantities of RAC proteins were mixed with 2× sample buffer (5% SDS, 5%
-mercaptoethanol, 400 mM Tris, pH 6.8, 10% sucrose), heated to 65°C for 5 min, and
separated by SDS-PAGE. The gels were either stained with Coomassie
blue or electroblotted to nitrocellulose (Schleicher and Schuell, Keene, NH) in 10 mM CAPS (pH 11)/10% methanol (Mayfield et al., 1994
). Filters were treated with rabbit polyclonal antisera specific for RB38, RB47,
or RB60.
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Results |
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Multiple psbA 5' UTR Exist In Vivo
S1 mapping of the psbA mRNA from C. reinhardtii previously identified two 5' termini, one 90 nt upstream of the
start codon and a more pronounced terminus 36 nt from
the start codon (Erickson et al., 1984). Multiple 5' termini
in this assay may reflect multiple transcription start sites,
posttranscriptional cleavage events, or RNA modifications
or structural elements which result in poor hybridization
of the radiolabeled probe. We used primer extension analysis to examine the 5' termini of the psbA 5' UTR. A radiolabeled oligonucleotide complementary to the 5' coding region of psbA was annealed with total RNA prepared
from wild-type C. reinhardtii. Reverse transcriptase was
used to extend the probe to the 5' terminus of the message. Extension products were separated by PAGE and quantified by PhosphorImager analysis. As shown in Fig.
1, 95% of the psbA message contains a 5' terminus 36 nt
upstream of the start codon whereas the remaining psbA
mRNAs feature 5' UTRs of 90 nt in length. Similar results
were observed previously by primer extension analysis
(Shapira et al., 1997
).
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The two predominant extension products could reflect different 5' termini or may indicate partial blocks in primer extension resulting from strong structural elements within the 5' UTR. To resolve this issue we used RNase H to cleave the psbA mRNA at the site of hybridization to a complementary oligonucleotide to generate 5' psbA fragments of a size that could be resolved by PAGE and subsequently visualized by Northern analysis. The size of the wild-type psbA 5' UTR can be compared with the 5' UTR from the hf261 strain, a nuclear mutant in which only the 90-nt psbA 5' UTR was observed by primer extension analysis (see Fig. 6). As shown in Fig. 2, the 5' fragment of the psbA message from hf261 is longer than the predominant wild-type psbA 5' fragment, confirming that the primer extension products reflect 5' UTRs of differing size. Therefore, the vast majority of wild-type psbA transcripts from C. reinhardtii begin 36 nt upstream of the start codon, adjacent to the RBS (Fig. 3 A).
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Neither primer extension analysis nor Northern analysis
can distinguish between 5' termini generated by transcription initiation and termini generated by posttranscriptional mRNA processing. However, the data will show
that the appearance of the 36-nt 5' UTR is most closely associated with posttranscriptional events, suggesting that
this terminus most likely results from mRNA processing. This conclusion is further supported by the observation
that partial deletion of a putative promoter element required for transcription initiation of the of the 36 terminus (Erickson et al., 1984
) had no affect on the appearance
of the 36-nt 5' UTR (Loop-del [Mayfield et al., 1994
]; data
not shown).
Processing Is Independent of the Primary mRNA Sequence at the Cleavage Site
To identify RNA elements required for processing, site-specific mutations were engineered within the 5' UTR of
the psbA gene which was then reintroduced into the C. reinhardtii chloroplast genome by particle bombardment (Mayfield et al., 1994). One of these constructs, Alter, replaced
the four uridine nucleotides just upstream of the RBS with
adenines (Fig. 3 B). In addition to disrupting the stem-loop element, this mutation changed the primary sequence
at the processing site. This mutation results in a 95% reduction in D1 protein synthesis and an 80% reduction in
D1 protein accumulation (Mayfield et al., 1994
). However,
primer extension analysis of total RNA isolated from
this strain demonstrates that cleavage of the psbA 5' UTR
is identical in extent and location to processing in wild-type C. reinhardtii (Fig. 4), indicating that processing is
not a function of the primary RNA sequence at the cleavage site.
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Processing of the psbA 5' UTR Is Dependent upon the Presence of a Competent and Accessible RBS
Many chloroplast-encoded mRNAs have a sequence resembling a prokaryotic-like SD that serves as an RBS.
However, because these sequences tend to vary in size and
location relative to their Escherichia coli counterparts, the
relevance of these RBSs in chloroplasts has been questioned (Fargo et al., 1998). Deletion of a putative SD sequence in the 5' UTR of the C. reinhardtii psbA mRNA
(RBS-del) eliminated psbA mRNA polyribosome association (Yohn et al., 1996
) and D1 synthesis in vivo (Mayfield
et al., 1994
), suggesting that this sequence is a functional
RBS. Additional mutations designed to disrupt the RBS
have been introduced into the psbA 5' UTR (Fig. 3 B).
These mutations either eliminated the RBS (RBS-del,
RBS-Alt), sequestered the RBS in an extended stem-loop
structure (RBS-paired), or changed the location of the RBS by replacing the wild-type RBS with another RBS
placed upstream of the stem-loop element (RBS-upstream).
All of these changes block ribosome association and abolish D1 protein synthesis in vivo, providing further evidence that this SD sequence functions as an authentic
RBS (Mayfield et al., 1994
; Bruick, R.K., and S.P. Mayfield, unpublished results). Total RNA was prepared from each of these mutants and primer extension analysis was
used to characterize the processing of the psbA 5' UTR.
As shown in Fig. 5, each of these mutants possess only one
5' terminus corresponding to the unprocessed psbA 5'
UTR. These data show that a functional RBS, required for
ribosome association and psbA translation, is also required for normal processing of the 5' UTR.
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Though processing of the psbA 5' UTR requires a SD sequence, and presumably initial ribosome association, processing is not dependent upon D1 protein synthesis. For example, a deletion within the psbA 5' UTR that positions the RBS 11 nt upstream of the start codon (RBS-11, Fig. 3 B) reduces psbA association with large polyribosomes and eliminates D1 protein synthesis in C. reinhardtii while enhancing translation of the same message in E. coli (Bruick, R.K., and S.P. Mayfield, manuscript in preparation). As demonstrated by a primer extension assay, the psbA 5' UTR from this mutant is still cleaved to the same extent and in the same position relative to the RBS as in the wild type (Fig. 5). The exact location of the processing site was confirmed by RNA sequencing (data not shown). Thus, we imagine that the RBS in this construct remains accessible for recognition by factors involved in the early assembly of a translation initiation complex before processing but that this mutant RBS lacks the ability to specify the correct initiation codon.
Processing of the psbA 5' UTR Is Reduced in Nuclear Variants Deficient in D1 Expression
Several nuclear mutations have been characterized in C.
reinhardtii that specifically affect expression of the D1
protein (Yohn et al., 1996; Yohn et al., 1998b
). In each of
these mutants (F35, hf149, hf233, hf261, hf859, and hf1085),
D1 protein synthesis is specifically lacking. While the primary defects of these mutations are not known, psbA association with polyribosomes is reduced or absent in the majority of these strains, suggesting that these mutations
affect translation initiation (Yohn et al., 1996
; Yohn et al.,
1998b
). Total RNA was prepared from each of these mutants and primer extension analysis of the psbA 5' UTR
was performed (Fig. 6). Each of these nuclear mutations
has a pronounced effect on the amount, but not the position, of processing of the psbA 5' UTR. In each case, the
ratio of transcripts featuring a 90-nt 5' UTR (
90) to processed transcripts (
36) is increased compared with the
wild type, with some mutants lacking any detectable psbA terminus 36 nt upstream of the initiation (hf261, hf1085).
Just as these nuclear mutants specifically affect translation of the psbA mRNA, the effect on mRNA processing
also appears to be psbA specific. In addition to the psbA 5'
UTR, the 5' UTR of the psbD mRNA (encoding the D2
protein) is also processed in C. reinhardtii (Rochaix et al.,
1984). Primer extension analysis using a radiolabeled oligonucleotide complementary to the psbD 5' coding region
was performed on total RNA prepared from the wild type
and the hf261 strain (Fig. 6). Whereas processing of the
psbA 5' UTR is completely absent in hf261, processing of
the psbD 5' UTR remains unaffected in this mutant as
does D2 protein synthesis (Yohn et al., 1998b
).
Processing of the psbA 5' UTR Is Not Dependent upon Illumination
To determine if the psbA 5' UTR is differentially processed in response to environmental conditions, total RNA
was isolated from C. reinhardtii grown under continuous
light or continuous darkness. Primer extension analysis indicated no difference in the relative amounts of the 5' termini of the psbA mRNA in response to growth under continuous light or in constant darkness (Fig. 7). A previous
study reported no change in psbA processing throughout the 18 h after a shift from low light to high light growth
conditions (Shapira et al., 1997). These results suggest that
cleavage of the psbA 5' UTR is not directly related to
light-dependent translational regulation.
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Processing of the psbA 5' UTR Is Not Dependent upon mRNA Association with Large Polyribosomes
During growth under constant light, only 35% of psbA
mRNA is associated with large polyribosomes (Yohn et al.,
1996). Polyribosome-associated RNA was isolated and
processing of the psbA 5' was analyzed by primer extension analysis. The extension products of psbA mRNA associated with polyribosomes were indistinguishable from
those observed in total RNA (Fig. 7). Because the D1 protein is inserted into the membrane during, or soon after,
synthesis, we examined the subset of polyribosome-associated psbA mRNA associated with thylakoid membranes.
If this subset of mRNAs comprises the pool of functionally
relevant psbA message, we might observe a difference in the
amount of processed psbA 5' UTR in this fraction as compared with total mRNA. Again, primer extension analysis
indicated no difference in the relative amount of processed
psbA 5' UTR between membrane-associated mRNA and
other RNA populations (Fig. 7).
Removal of the Stem-Loop Element from the psbA 5' UTR Does Not Affect Binding of the Light-regulated Protein Complex In Vitro
The stem-loop element upstream of the RBS had been
identified previously as a binding site for a protein complex involved in the dynamic regulation of psbA translation in response to light (Danon and Mayfield, 1991).
However, as a consequence of processing, >95% of the
steady-state levels of psbA message lacks this stem-loop element in vivo. To determine if the processed psbA 5'
UTR was still capable of being recognized by the protein
complex, a gel mobility shift experiment was used to compare complex binding to the two psbA 5' UTRs in vitro.
Heparin-agarose purified proteins from wild-type C. reinhardtii were incubated with in vitro transcribed RNAs corresponding to either the 90-nt psbA 5' UTR (
90) or the processed 5' UTR (
36). Complex binding is reflected by
the appearance of an RNA-protein complex with reduced
mobility in a nondenaturing polyacrylamide gel. As shown
in Fig. 8, both RNAs are bound by protein. Furthermore,
each RNA is able to compete the binding of the complex
to the other RNA when added in excess as a cold competitor of the labeled RNA for the protein complex. The possibility of the
90 RNA being processed to the
36 RNA
in vitro before complex formation is excluded by the observation that the migration of the
90 RNA-protein
complex is retarded relative to the
36 RNA-protein
complex.
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Previous experiments have identified the primary psbA
binding protein to be RB47, the cPABP (Danon and Mayfield, 1991; Yohn et al., 1998a
). UV cross-linking of both
radiolabeled transcripts (
90 or
36) complexed with
heparin-agarose purified proteins resulted in the specific
labeling of RB47 (Fig. 9). The lower intensity of RB47 labeling using the
36 transcript may be due to fewer radiolabeled residues in close proximity to the bound cPABP
for the shorter RNA fragment or may reflect slightly lower binding affinity of the 36-nt 5' UTR compared with the
90-nt 5' UTR.
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RAC was used to further characterize complex binding
to the processed 5' UTR. RNA transcripts corresponding
to the entire (90) and processed (
36) psbA 5' UTRs
were immobilized on solid supports. Heparin-agarose-
purified C. reinhardtii lysate was passed over each support
and bound proteins were eluted with high salt. An identical set of proteins was eluted from both columns as observed by Coomassie blue staining (Fig. 10 A). Western
analysis with antibodies specific for the cPABP (RB47),
the chloroplast-localized protein disulfide isomerase (RB60)
(Kim and Mayfield, 1998
), and a 38-kD component of the
psbA binding complex (RB38) further demonstrated that
both psbA 5' UTRs are recognized by an identical protein complex in vitro (Fig. 10 B). Taken together, these results
predict that removal of the stem-loop element from the
psbA 5' UTR does not prevent the binding of the psbA-specific protein complex nor does it preclude the dynamic
regulation of translation in response to light.
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Discussion |
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In both C. reinhardtii and higher plants, the expression of
nuclear and chloroplast genes is tightly regulated to allow
for a rapid response to fluctuations in environmental conditions. The regulation of chloroplast-encoded genes is dependent upon sequences within their 5' UTRs that influence message stability, translation, and possibly message
localization (Gillham et al., 1994; Mayfield et al., 1995
;
Rochaix, 1996
). Mutational analysis of the 5' UTR of the
psbA mRNA has shown that RNA elements, including a
consensus SD sequence and an upstream stem-loop element, mediate D1 expression (Mayfield et al., 1994
). In
this study, primer extension analysis of the psbA mRNA
from a variety of C. reinhardtii mutants has revealed a correlation between processing of the 5' UTR and association
of the mRNA with ribosomes. Alterations to the psbA 5'
UTR that disrupt the RBS prevent RNA processing. Nuclear mutations that specifically block D1 protein synthesis, in part by reducing ribosome association, specifically
reduce psbA mRNA processing. The proximity of the processing site to an accessible and competent RBS, and the
influence on processing of nuclear-encoded factors that affect ribosome association, suggest a relationship between ribosome association and processing of the 5' UTR of the
psbA mRNA.
In contrast to psbA messages from C. reinhardtii, generally only one 5' terminus, corresponding to the unprocessed 5' UTR from C. reinhardtii, has been observed for
the psbA mRNA isolated from higher plants (Zurawski
et al., 1982; Link and Langridge, 1984
; Sugita and Sugiura,
1984
; Boyer and Mullet, 1986
; Boyer and Mullet, 1988
;
Hanley-Bowdoin and Chua, 1988
; Liere et al., 1995
). Nevertheless, processing of mRNAs is a common phenomenon in higher plant chloroplasts, in which most messages
are transcribed as polycistronic transcripts. For example,
the petD mRNA, which is transcribed as a dicistronic message downstream of petA, contains elements within the 5'
UTR that are required for accumulation of the monocistronic petD mRNA. Failure to process the petD 5' UTR
results in the loss of translation of the petD message
(Sakamoto et al., 1993
; Sakamoto et al., 1994a
,b).
Processing of the 5' UTR of the rbcL transcript, the
chloroplast mRNA encoding for the large subunit of
Rubisco, is also correlated with ribosome association and
protein synthesis. In C. reinhardtii, for example, changes in
large subunit synthesis in response to light levels coincide
with changes in processing of the 5' UTR (Shapira et al.,
1997). rbcL transcripts with longer 5' UTRs (beginning 168 nt upstream of the start codon rather than 93 nt) are
not associated with polyribosomes and probably are not
translationally competent. Recovery of large subunit synthesis is accompanied by an increase in the relative level of
processed mRNAs. In mature barley leaves, inhibition of
large subunit protein synthesis by methyl jasmonate is accompanied by a change in the predominant rbcL 5' UTR,
resulting from alternative processing of the primary transcript. Once again, the longer 5' UTR is not associated
with polyribosomes (Reinbothe et al., 1993
). These observations further suggest that in both C. reinhardtii and
higher plants, processing of 5' UTRs coincides with ribosome association of those mRNAs. Thus, although it is often assumed that 5' end formation occurs before, and is
necessary for proper translation initiation, the available
data also support the possibility that processing of mono-
and polycistronic 5' UTRs may be a consequence of ribosome association, rather than a prerequisite for it.
Although processing of the C. reinhardtii psbA 5' UTR appears to be linked with translation initiation at the level of small ribosomal subunit recognition of the SD sequence association, no correlation was observed between processing and the dynamic regulation of translation in response to illumination. When C. reinhardtii is grown in the dark rather than the light, D1 protein synthesis is reduced in conjunction with reduced binding activity of the cPABP complex. Nevertheless, no difference in processing was observed between psbA mRNA isolated from C. reinhardtii grown in the light or in the dark, nor is there a direct linear relationship between the amounts of light-regulated translational activator proteins, such as the cPABP, and processing in the nuclear mutants. Therefore, processing is dependent upon at least the initial stages of ribosome assembly at the SD sequence, which occurs before, and independent of, dynamic light-dependent translation. However, some proteins may be common to both of these processes.
Previous studies have demonstrated that mutations introduced within the psbA 5' UTR upstream of the RBS
can disrupt D1 expression. Typically, these mutants accumulate normal amounts of psbA mRNA but synthesize D1
protein at <5% of the wild-type level (Mayfield et al.,
1994). Nevertheless, the psbA 5' UTR in each of these mutants is processed normally. Processing within these transcripts removes the mutated RNA upstream of the RBS
and results in a steady-state pool of psbA transcripts that is
virtually indistinguishable from the wild type. Therefore,
sequences upstream of the RBS must influence D1 expression before processing and ribosome association, possibly by acting on steps such as formation of a preinitiation
complex or perhaps even in localization of the psbA
mRNA to specific regions of the chloroplast (Rochaix,
1996
). The D1 protein must be synthesized in close proximity to the thylakoid membrane, and translational activators, including the cPABP, have been shown to be associated with a specific membrane fraction within the chloroplast
(Zerges and Rochaix, 1998
). Perhaps these upstream sequences act in concert with translational activator proteins
to target the psbA mRNA to membrane-associated ribosomes.
Sequences upstream of the processing site could also influence chloroplast gene expression at the level of message
stability, acting before the early stages of ribosome association. For example, the 5' UTR of the chloroplast psbD
mRNA from C. reinhardtii is processed in a manner similar to that of the psbA mRNA (Rochaix et al., 1984; Erickson et al., 1986
; Nickelsen et al., 1994
). Analysis of nuclear
mutants that destabilize the psbD mRNA has indicated
that the sequences upstream of the processing site in the
psbD 5' UTR may mediate this mRNA stability by interacting with nuclear encoded factors (Nickelsen et al.,
1994
). Furthermore, a deletion engineered within the
psbD 5' UTR designed to initiate psbD transcription at
the processing site resulted in a lack of psbD mRNA accumulation despite a wild-type rate of transcription of the
mRNA (Rochaix, 1996
). An analogous chloroplast deletion engineered to initiate psbA transcription at the processing site also resulted in failure to accumulate any psbA
mRNA (data not shown). These data show that sequences
upstream of the RBS can influence message stability,
likely acting during the early stages of mRNA maturation.
Interestingly, an endonucleolytic cleavage site associated
with mRNA degradation has been mapped within the spinach psbA 5' UTR in vitro. Similarities exist between
this endonucleolytic cleavage site and the processing site
within the C. reinhardtii psbA 5' UTR. Both cleavage sites
are located immediately upstream of a consensus RBS. In
spinach, this site also defines the upstream boundary of the
core RNA element recognized by an RB complex that includes the ribosomal protein S1 (Klaff, 1995
; Alexander et
al., 1998
). The close proximity of this cleavage site in spinach
psbA mRNA to the putative RBS and the site of ribosome
assembly may reflect an intimate relationship between translation initiation, mRNA processing, and message stability. This relationship is also observed for psbA mutants in C. reinhardtii (RBS-del, RBS-Alt, RBS-paired) in which the loss
of ribosome binding due to the absence of a SD sequence
results in the absence of processing and a reduction in
psbA mRNA accumulation (Mayfield et al., 1994
).
Originally, mutations to the upstream stem-loop element of the psbA mRNA were thought to influence D1
translation by altering the binding site of the light-regulated cPABP complex as the stem-loop element was found
to be protected by protein binding in a T1-gel mobility
shift assay (Danon and Mayfield, 1991; Mayfield et al., 1994
). However, gel shift assays, UV cross-linking, and
RAC assays presented here all indicate that both the 90-nt
and processed psbA 5' UTRs are recognized by an identical set of psbA-specific RB proteins. Thus, dynamic light-regulated translation via binding of these activator proteins should still be possible with the truncated psbA
mRNA.
Based on these and other results, we propose a model
for psbA maturation and translation in the C. reinhardtii
chloroplast. The psbA mRNA is transcribed with a 5'
UTR extending at least 90 nt upstream of the start codon.
The 5' UTR is rapidly processed to the 36 transcript but
not before the upstream RNA elements influence D1 expression. These upstream elements could affect expression
at a number of key steps including RNA stability, formation of a preinitiation complex, or mRNA localization.
Processing is mediated by proteins, including ribosomal
subunits and possibly other initiation factors, that recognize the RBS during the early stages of ribosome association. The nuclease responsible for processing has not yet
been identified but appears to be closely associated with a
ribosomal subunit or with a protein complex required for ribosome association. Interaction of the ribosomal subunits and associated proteins with the processed 5' UTR
may act to stabilize the message. Processing may also initiate an mRNA degradation pathway affecting the half-life
of the transcripts. Whereas removal of the upstream sequences may be necessary to make the message competent
for high levels of translation, processing alone is not sufficient for translation of the message as evidenced by mutations, both chloroplast and nuclear, that affect psbA translation without eliminating processing. Upon illumination,
the binding activity of a set of psbA-specific RB proteins,
including the cPABP, is enhanced in response to increased
photosynthetic activity. Binding of this protein complex
stimulates translational activity, probably acting via recognition of the psbA 5' UTR through a stretch of adenine
residues between the RBS and the start codon (Bruick, R.K., and S.P. Mayfield, manuscript in preparation).
While the molecular basis underlying each of these events
remains unknown, we have begun to define distinct RNA
elements within the psbA 5' UTR that are recognized by
trans-acting factors to orchestrate message stability, mRNA
processing, general translational competency, and dynamic
translational activation.
![]() |
Footnotes |
---|
Address correspondence to Stephen P. Mayfield, Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (619) 784-9848. Fax: (619) 784-9840. E-mail: mayfield{at}scripps.edu
Received for publication 10 August 1998 and in revised form 21 October 1998.
We thank Christopher Yohn and Amybeth Cohen for critical reading of this manuscript and for providing membrane-associated RNA (A. Cohen).
This work was supported by funds from the National Institutes of Health to S.P. Mayfield (GM54659). R.K. Bruick was supported by a National Science Foundation (NSF) predoctoral fellowship and an NSF Graduate Research Traineeship in Macromolecular Structure and Plant Biology.
![]() |
Abbreviations used in this paper |
---|
cPABP, chloroplast poly(A)-binding protein; nt, nucleotide; RAC, RNA affinity chromatography; RB, RNA-binding; RBS, ribosome binding sequence; SD, Shine-Dalgarno; UTR, untranslated region.
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