From the Department of Biological Sciences, State University of New York, Buffalo, New York 14260
Received for publication, October 10, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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In amaranth, a C4
dicotyledonous plant, the plastid rbcL gene (encoding the
large subunit of ribulose-1,5-bisphosphate carboxylase) is regulated
post-transcriptionally during many developmental processes, including
light-mediated development. To identify post-transcriptional regulators
of rbcL expression, three types of analyses (polysome heel
printing, gel retardation, and UV cross-linking) were utilized. These
approaches revealed that multiple proteins interact with 5' regions of
rbcL mRNA in light-grown, but not etiolated, amaranth plants. Light-associated binding of a 47-kDa protein (p47), observed by
UV cross-linking, was highly specific for the rbcL 5' RNA. Binding of p47 occurred only with RNAs corresponding to mature processed rbcL transcripts (5'-untranslated region (UTR)
terminating at Post-transcriptional processes play a key role in regulating the
expression of genes encoding photosynthetic enzymes such as
ribulose-1,5-bisphosphate carboxylase
(RuBPCase)1 (1-5). In
amaranth, a C4 dicotyledonous plant, additional layers of
regulation have been incorporated into the control of photosynthetic genes to achieve the specialized expression patterns required for this
modified photosynthetic pathway (6). Post-transcriptional alterations
in the expression of chloroplasts genes encoding the large subunit
(LSU, encoded by rbcL genes) and nuclear genes encoding the
small subunit (encoded by RbcS genes) of the amaranth
RuBPCase occur in response to numerous developmental, metabolic, or
environmental cues.
Two types of light-shift experiments show that rapid changes in
RuBPCase gene expression are induced in response to changes in
illumination, due to regulation occurring at two translational steps.
First, when amaranth seedlings grown in complete darkness (etiolated)
are transferred to light (light shift), RuBPCase synthesis is rapidly
induced at the level of translational initiation (7, 8).
rbcL mRNAs accumulate in the absence or presence of
illumination, but these associate with polysomes and are translated
only in the light. The light-mediated initiation of translation is
mostly specific for RuBPCase, since transcripts encoding
nonlight-regulated proteins are associated with polysomes regardless of
the light conditions. Second, when light-grown seedlings are
transferred to complete darkness (dark shift), synthesis of both
RuBPCase subunits is rapidly repressed (7, 9), even though both
transcripts remain in association with polysomes. These findings
indicate that during the dark shift, translation of both RuBPCase
subunits is regulated at the elongation step.
Post-transcriptional regulation of rbcL and RbcS
gene expression has also been implicated during the initial stages of
amaranth C4 leaf development (10) and in association with
changes in photosynthetic activity (11). For the plastid-encoded
rbcL gene, post-transcriptional control of differential
rbcL mRNA accumulation in isolated bundle sheath (bs)
and mesophyll (mp) chloroplasts of mature amaranth leaves has been
demonstrated, apparently acting through differential mRNA stability
in the two plastid types (12).
There are many examples of plastid genes in plants and algae that are
regulated at the levels of translation, control of mRNA stability,
or RNA processing (8, 9, 13-22). In some cases, nuclear-encoded
trans-acting proteins have been shown to interact specifically with
cis-acting regions within the 5'- or 3'-UTRs of the plastid mRNAs,
and these have been implicated in the regulation of one or more of
these processes (19, 23-32). A well characterized example of a
plastid-encoded mRNA that is regulated by light at the level of
translation is psbA, which encodes the D1 protein of
photosystem II. In plastids of tobacco, spinach, and
Chlamydomonas, changes in psbA translation have
been correlated with binding of a protein complex at the 5'-UTR (23,
28, 29). An inverted repeat sequence within the Chlamydomonas
psbA 5'-UTR interacts directly with a psbA-specific
47-kDa protein (a plastid form of poly(A)-binding protein). This
binding is affected by redox potential and by interactions with an
associated 60-kDa protein disulfide isomerase (23, 32-35).
Little is known about control regions of the plastid-encoded
rbcL transcripts or factors that interact with these
regions, even though this gene encodes one of the most essential,
abundant, and highly regulated proteins of photosynthesis. It has been
shown that rbcL 5'-UTR can affect both translation rates and
the stability of the transcript in plastids of Chlamydomonas
(36) and tobacco (26, 37), causing enhanced translation and more rapid
degradation of a chimeric RNA in the light relative to the dark. The 5'
portion of the coding region also appears to be involved in stabilizing the transcript (36). In Euglena chloroplast extracts,
modifications to the rbcL 5'-UTR near the start codon,
either deletions or the addition of secondary structure, reduce rates
of translational initiation (38, 39). In plastids of a higher plant,
barley, methyl-jasmonate-induced processing of the rbcL
5'-UTR appears to play a role in regulating translation of the LSU
polypeptide (40).
To identify components required for post-transcriptional
rbcL regulation in the chloroplasts of a C4
plant, we examined and characterized proteins capable of binding to the
amaranth rbcL transcripts in response to changes in
illumination. Because sequences involved in protein binding and
post-transcriptional regulation have been identified within the 5'
regions of several mRNAs (1-4), we concentrated on RNA/protein
interactions occurring at or near the 5'-UTR. We show that specific,
light-associated protein binding occurs in vivo and in
vitro, and this binding correlates with light-mediated changes in
LSU synthesis in both leaves and cotyledons. In addition, we
demonstrate that binding of a 47-kDa protein is dependent on the length
of the 5'-UTR, occurring only with RNAs containing 5' ends that
correspond to mature rbcL transcripts. These results
indicate that regulatory regions within or near the rbcL
5'-UTR interact with trans-acting binding proteins in a
light-dependent manner, and that differential 5' mRNA
processing in vivo may work in coordination with light
activation of protein binding to regulate rbcL synthesis in
the chloroplasts of a C4 plant.
Plant Materials and Growth Conditions--
For light-grown
plants, seeds of Amaranthus hypochondriacus var. 1023 were
germinated and grown in a growth chamber (Conviron, Asheville, NC) at
24 °C with 14 h per day of illumination at an approximate intensity of 170-200 µE m
Bs and mp protoplasts were prepared from fully expanded green leaves
and separated by sucrose density gradient centrifugation as previously
described previously (12). Chloroplasts were isolated from the
separated protoplasts and purified on Percoll gradients as described
(12), and the separated plastids were used to prepare cell
type-specific soluble chloroplast extracts.
Polysome Isolation--
Polysomes used for heelprinting or for
sucrose gradient analysis were isolated from leaves or cotyledons of L,
E, or DS plants, as described previously (8, 9).
RNA Heelprinting--
Protein-protected RNA fragments were
generated by modification of the heelprinting procedure of Wolin and
Walter (41). Briefly, polysomes were treated with micrococcal nuclease
to degrade RNA not protected by protein. The nuclease was inactivated
by addition of EGTA, and the reactions were frozen at
For mapping the positions of protein-protected RNA fragments, the
fragments were first annealed to single-stranded pRbl-1 DNA template,
corresponding to the entire mRNA coding region of the amaranth
rbcL gene (42), and primer extension was performed. Each
annealing reaction contained 75 fmol of rbcL ssDNA, 150 fmol of 32P-end-labeled primer, and 1 µg of protected RNA
fragments in hybridization buffer (33 mM Tris acetate, pH
7.7, 67 mM potassium acetate). The reactions were heated to
65 °C for 5 min and then slowly cooled to 30 °C for ~30 min.
After annealing, extension reactions were carried out in the same
buffer, with 20 mM magnesium acetate, 0.334 mM
of each dNTP, 1 mM dithiothreitol, and 3 units of T4 DNA
polymerase. After 30 min at 37 °C, the extension products were
extracted with phenol/chloroform/isoamyl alcohol, precipitated with
ethanol, and resuspended in loading buffer (95% formamide, 10 mM EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol). The
samples were heated to 70 °C for 5 min and fractionated on 8.3 M urea, 6% polyacrylamide gels, together with sequencing
reactions of the pRbl-1 clone to map the locations of the protected fragments.
Preparation of Chloroplast-soluble Protein
Extracts--
Chloroplast extracts for binding assays were prepared
from leaves or cotyledons according to published protocols (27, 43). Briefly, total soluble proteins were prepared from a chloroplast lysate
that had been clarified by centrifugation at 175,000 × g for 3 h. The clarified supernatant was then passed
over a DE52-cellulose anion exchange column, and the flow-through was
precipitated with ammonium sulfate at 60% saturation. Precipitated
protein was pelleted at 80,000 × g in an SW 41 rotor for 30 min. The supernatant was discarded, and the pellet
was resuspended in Buffer E containing 20 mM HEPES, pH 7.9, 60 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 2 mM dithiothreitol, and 17%
glycerol. Soluble proteins in Buffer E were loaded onto a
heparin-agarose column (Sigma) equilibrated with Buffer E. The sample
was passed over the column twice, eluted with Buffer E, and the
flow-through collected. Protein concentrations were determined by using
a Bradford protein assay (Bio-Rad).
Generation and Preparation of 32P-Labeled rbcL 5' RNA
Transcripts--
DNA fragments used for in vitro synthesis
of rbcL 5' RNAs were polymerase chain reaction-amplified
from pRBL1, using primers to specific regions of the UTR (42). Primers
at the 5' end contained a T7 promoter. RNA corresponding to various
regions of the rbcL 5' RNA were synthesized in
vitro in 20-µl reactions containing 1 µg of DNA template, 10 mM dithiothreitol, 1 mM ATP, CTP, and GTP, 500 µM UTP, 100 µCi of [ Gel Mobility Shift Assays--
Gel mobility shift assays were
performed according to the methods of Chen et al. (44),
except without the addition of RNase T1. Competition analyses were
performed using unlabeled rbcL RNA as self-competitor or
heterologous yeast viral RNA (7z-AS, a 130-nt 3'-UTR sequence from a
yeast double-stranded RNA virus, generously provided by Dr. J. Bruenn,
State University of New York, Buffalo) (45). The probe and the
unlabeled competitor RNAs were added to the reactions prior to the
addition of the protein extracts, or protein was added to the unlabeled
competitor and preincubated before addition of the labeled 5' RNA.
Identical results were obtained from both types of competition
reactions. Gels were visualized using a PhosphorImager equipped
with ImageQuant version 4.2 software (Molecular Dynamics, Sunnyvale, CA).
UV Cross-linking Assay--
UV cross-linking experiments were
performed according to published methods (28, 46) with some minor
modifications. In all experiments, unless otherwise noted, 10 µg of
protein extract was incubated with 15 fmol (~1.5-2.5 × 105 total cpm) of in vitro transcribed
radiolabeled RNA in a 20-µl reaction volume containing 40 mM KCl, 10 mM MgCl2, 3 mM dithiothreitol, 0.05 mM EDTA, 8.5% (v/v)
glycerol, 10 mM HEPES, pH 7.9, and at least 10 µg of tRNA
as a nonspecific competitor. Competition experiments with excess
unlabeled RNAs were performed as described for gel mobility assays
(relative concentrations of the competitors are detailed in the figure
legends). After incubation for 10 min at 25 °C, open
microcentrifuge tubes containing the binding reactions were
irradiated for 30 min with UV light (254 nm) using a UV cross-linker (Fisher) set to an energy level of 0.18 J/cm2 at 25 °C.
The RNA was digested with 20 units of RNase T1 and 1 µg of RNase A
per sample at 37 °C for 30-45 min. For SDS-gel electrophoresis, 1 volume of 8% SDS, 3 M Primer Extension and DNA Sequencing Reactions--
A primer
corresponding to the first 15 nt of coding region of the
rbcL transcript (42) was 5' end-labeled with 32P
using polynucleotide kinase, hybridized to total RNA (isolated from
tissues of plants grown under various illumination conditions), and
extended using SuperScript II (Life Technologies, Inc.). The extension
reactions were phenol/chloroform/isoamyl alcohol-extracted and
precipitated with ethanol overnight. The reactions were resuspended, boiled, and loaded onto 6% polyacrylamide urea gels for analysis. 32P-Labeled sequencing reactions were prepared using
Sequenase (Amersham Pharmacia Biotech) with pRbl-1 DNA as a template
and run in adjacent lanes to determine the size of the extensions. Gels
were analyzed using a PhosphorImager.
Analysis of Polysome-associated Transcripts--
Polysomes
isolated from L seedlings were separated on 10-50% sucrose gradients
according to methods described previously (8, 9). Gradient profiles
were determined using an ISCO model 185 density gradient fractionator
and model UA-5 absorbance/fluorescence detector. RNA was isolated from
the gradient fractions (8) and analyzed by primer extension analysis as
described above.
In Vivo Protein-protected RNA Fragments on rbcL
Transcripts--
The amaranth RuBPCase LSU polypeptide is synthesized
only in light-grown (L) plants and not in dark-grown (etiolated, E) or dark-shifted (DS) plants. RNA heelprinting analysis was undertaken to
determine whether there are corresponding light-associated differences
in RNA-protein interactions occurring in vivo within the
5'-UTR or other 5' regions of the rbcL transcript.
Polysome-associated RNAs purified from plants incubated under various
light conditions were subjected to nuclease digestion, such that
regions of the RNA that are associated with protein are protected. By
visualizing the positions of protected RNA fragments, sites of
RNA-protein interactions, as they occur in vivo, are revealed.
Fig. 1 indicates the locations of several
protected RNA fragments produced by the heelprinting reactions. Most of
these originated from within the 5'-UTR of the rbcL
mRNA, between the 5' terminus of the mature transcript at
Several protected RNA fragments originated from within the coding
region as well. One of these (*, lane L) occurred only in polysomal RNAs from L plants, and others occurred in RNAs from both L
and DS plants (lanes DS and L). As with the
5'-UTR fragments, none of these coding region fragments occurred in
RNAs isolated from E plants.
For Fig. 1, all of the RNase-protected fragments that were present in
the polysomal RNA lanes, but not in the control lane (Fig. 1,
lane C), were eliminated by treatment with protease (data not shown). Therefore, the protected RNA fragments originating at sites
within the rbcL 5'-UTR and within the open reading frame were dependent on RNA-protein interactions.
Identification of Light-associated rbcL 5' RNA Binding Protein
Activity by Gel Mobility Shift (GMS)--
Soluble chloroplast protein
extracts from L or E plants were purified by heparin-agarose
chromatography to enrich for nucleic acid binding proteins and
incubated with in vitro synthesized 32P-labeled
rbcL 5' RNA (corresponding to the 5' end of mature processed rbcL RNA, terminating at Identification of Individual Proteins Binding rbcL 5' RNA by UV
Cross-linking--
When 32P-labeled 5' RNA was incubated
with extract from L or E plants and UV cross-linked, there were a
number of binding proteins observed (Fig.
3A, 1st lane). Most notable
was a doublet protein band migrating at ~47 kDa (p47). When the
rbcL 5' RNA was incubated with E extracts, no significant
protein cross-linking to the RNA was observed (Fig. 3A, 3rd
lane), indicating that p47 is a light-associated RNA-binding
protein.
p47 binding activity in L extracts was usually observed as a doublet
band. This could be due to modified forms of a single protein or two
separate proteins of similar size that bind to rbcL 5' RNA.
For the studies described here, p47 will be referred to as a single
protein. The p47 doublet band was not observed in control reactions
cross-linked in the absence of any protein extract (data not shown) or
with L extracts in the presence of excess self-competitor (Fig.
3A, 2nd lane). The appearance of the light background smear
in some competitor lanes shown here and in the following figures is due
to incomplete RNase digestion of the radiolabeled RNA, in the presence
of excess amounts of unlabeled transcript.
The specificity of rbcL 5' RNA interactions with p47 in L
extracts was addressed by competition assays (Fig. 3B).
Self-RNA competed for cross-linking to p47 (as well as some minor
bands) at 100× molar excess, and competition was complete by 10,000×. As with the GMS results, 7z-AS RNA did not compete for cross-linking even at very high molar excess (10,000× or greater). In addition, the
homopolymers poly(A), poly(U) (not shown), and the 5'-UTR of
psbA did not compete for binding at even the highest
concentrations. These findings indicate that the light-associated
binding of p47 is specific to the rbcL 5' RNA.
Protein Binding to rbcL 5' RNA Is Dependent on the Length of the
5'-UTR--
In dicots, rbcL mRNA is transcribed from a
single highly conserved promoter region located 156-185 nt upstream of
the start codon (47). The primary transcript is constitutively
processed such that the 5' end is within the range of
The effects 5'-UTR length on p47 binding were examined by using
rbcL 5' RNAs containing different regions of the 5'-UTR
(Fig. 4A). The following RNAs
had 5' termini occurring at different sites within the 5'-UTR and also
contained rbcL coding region to +60. These probe RNAs were
As shown in Fig. 4B, the predominant doublet band at 47 kDa
was observed only with the rbcL 5' RNA terminating at
To determine whether differences in the length of the amaranth
rbcL 5'-UTR occur in vivo, primer extension
analysis was used. A primer corresponding to the 5' end of the
rbcL coding region was 5' end-labeled and used in primer
extension analysis with equalized amounts of total RNA isolated from L
and E plants. Fig. 3C shows that there were two prominent 5'
ends observable in both RNA samples as follows: one at In Vivo Differences in 5'-UTR Ends Correlate with Differences in
Binding of p47 in Vitro--
Fig. 4D shows that only one of
three RNAs corresponding to in vivo rbcL mRNAs, the
Fig. 4 indicates that in L plastid extracts, sequences required for p47
binding occur within a 52-nt region of the 5'-UTR, between The 5'-UTR and Coding Region Are Required for p47 Binding--
To
determine whether the 5'-UTR alone is sufficient for p47 binding, probe
RNAs were prepared that contained only the UTR portion of the
rbcL 5' RNA (from +1 to Differences in rbcL 5'-UTR Length Do Not Correlate with Changes in
Polysome Association--
Since p47 binding correlates with
light-mediated activation of rbcL gene expression, and
binding is not detected to rbcL mRNAs with 5'-UTRs
extending beyond
In addition to the three most prominent 5' rbcL extension
products, Fig. 6B shows that there were also some less
abundant products on polysomes, notably those terminating at p47 Binding and Patterns of rbcL Gene Expression--
Possible
correlations between 5' RNA binding activity and previously described
post-transcriptional changes in rbcL gene expression were
investigated using plastid extracts isolated from plants grown under
different conditions of illumination or from separated bs and mp
plastids (Fig. 7). UV cross-linking
analysis revealed the typical p47 doublet band in extracts prepared
from leaves as well as cotyledons of L plants (Fig. 7, light
and 6-day light cots, respectively), both of which function
as photosynthetic tissues in amaranth seedlings (49). p47 binding was
also apparent in DS plants (Fig. 7, 6-day dark-shift cots)
but not in etiolated tissues (Fig. 7, etiolated).
Protein extracts from Percoll-purified chloroplasts that had been
isolated from separated bs and mp protoplasts were used to determine
whether differential binding of p47 correlates with post-transcriptional differences in rbcL mRNA
accumulation occurring in the two plastid types (12). As shown in Fig.
7 (Bundle sheath and Mesophyll lanes), binding
patterns for both of the highly purified plastid protein extracts were
similar, although intensity of the cross-linked p47 band was
approximately 4-fold lower in the mp extracts than in the bs extracts.
Is p47 Similar to Other Known Chloroplast 5' RNA-binding
Proteins?--
Although binding of p47 to amaranth rbcL RNA
was not competed with amaranth psbA transcript, this binding
activity does share some properties with light-associated proteins that
interact with the 5'-UTR of psbA mRNA in other plant
systems (1-4). To distinguish the binding activities associated with
these two plastid-encoded transcripts, we compared patterns of UV
cross-linking to rbcL and psbA 5' RNAs in L
plastid extracts. As shown in Fig. 8, the protein cross-linking patterns to these two RNAs were distinct. The
psbA transcript cross-linked to two major proteins (Fig. 8, psbA lane1), one at ~48 kDa (just above the cross-linked
proteins observed with rbcL 5' RNA) and the other at
~41-43 kDa. These are similar in size to two psbA 5'-UTR
RNA-binding proteins identified in spinach (28, 50). A third weaker
band could also be seen at ~28-30 kDa. This distinct protein
cross-linking pattern was specific to the psbA 5' RNA, since
the cross-linking could be competed with unlabeled self-competitor
(Fig. 8, psbA, lane 2) but not with up to a 10,000× molar
excess of rbcL 5' RNA (Fig. 8, psbA, lane 3).
Based on their different sizes and specificities of binding, it is
evident that the major p47 rbcL 5' RNA-binding proteins are
distinct from those that interact with psbA 5' RNA in
amaranth chloroplasts.
In this study, three methods were used to identify amaranth
rbcL mRNA-binding proteins that associate with
rbcL 5' RNA only in plastid extracts from plants grown in
the light and not in the dark, conditions that produce significant
differences in synthesis of the LSU protein (6, 7). Light-associated
mRNA/protein interactions such as these, occurring at the 5'-UTR,
are potential candidates for regulators of rbcL translation,
processing, or stability in the plastids of this C4 plant.
Proteins Bound to the 5' Regions of Polysome-associated rbcL
mRNAs in Vivo--
RNA heelprinting analysis revealed several RNA
fragments that were protected from RNase digestion by protein
association. Most of these were from L plants or from DS plants (when
rbcL mRNAs are associated with polysomes (8, 9)) and
were greatly reduced or absent in mRNAs from E plants (when
rbcL mRNAs are not on polysomes (8)). The
RNase-protected fragments were isolated by centrifugation through two
sucrose cushions, and relatively small complexes (6 S or less) do not
pellet under these conditions (51). Thus any fragments identified by
this heelprinting assay must be associated with ribosomes or with a
large protein complex.
Two predominant fragments from within the 5'-UTR were associated only
with RNAs from L plants, where the rbcL mRNAs are
actively translated (8). One of these corresponded to the processed end
of the amaranth rbcL mRNA, and another was from an
AU-rich region at
In addition to the L-specific fragments, there were two easily detected
fragments located near the Shine-Dalgarno sequence that were present in
RNAs from L as well as DS plants but were not observed with the E RNAs.
Ribosome binding at the Shine-Dalgarno sequence of rbcL
mRNA from barley chloroplasts has been observed using toeprinting
analysis (52), and the protected fragments at the Shine-Dalgarno region
may represent ribosome binding at this site. Other 5' RNA fragments
detected in our assay occurred only in association with rbcL
mRNAs from L plants and are potential candidates for
light-dependent regulatory complexes.
Light-associated Protein Binding to the rbcL 5' RNA in
Vitro--
RNA heelprinting experiments show that there are in
vivo differences in the occurrence of large protein complexes
bound to specific 5'-UTR and coding regions of polysome-associated
rbcL mRNAs in L and E plants. Similarly, the in
vitro binding extracts indicated the presence of light-associated
protein binding that is specific to the rbcL 5' region. This
binding activity was visualized as a slow-migrating shifted band (5'
LRP) on nondenaturing gels and as a 47-kDa protein doublet (p47) by
direct UV cross-linking. Both binding activities occurred only in
extracts prepared from L plants and not from E plants. The very high
specificity of p47 for rbcL 5' RNA and its requirement for
RNA corresponding to fully the processed transcript distinguish it from
other plastid mRNA-binding proteins that have been found in
chloroplasts (19, 23, 28, 31, 53, 54).
Sequences necessary for p47 binding in L extracts occurred between
L versus D amaranth seedlings showed differences in the
length of the rbcL 5'-UTR, and in L plants all of the
rbcL transcripts (full-length, intermediate, and mature)
were found in association with polysomes that were presumably involved
in synthesis of the LSU protein. It is likely that the intermediate
length 5'-UTR transcripts result from post-transcriptional processing.
The single rbcL promoter and 5'-leader regions are highly
conserved among dicots, and there are no promoter-like sequences
located between the primary transcript and the shortest mature
transcript (47). The observation that intermediate processed
rbcL transcripts are polysome-associated indicates a
relationship between light-associated p47 binding, light-associated
transcript processing, and light-dependent rbcL
translation in amaranth chloroplast, although the nature of this
relationship remains to be determined. Other studies have linked
mRNA processing at the 5'-UTR with polysome association in
chloroplast. In barley, it has been shown that differential 5'-UTR
processing, induced by the plant growth regulator methyl-jasmonate, regulates translational initiation (40). In Chlamydomonas
plastids, ribosome association appears to be necessary for processing
of the psbA 5'-UTR (58). Unlike the amaranth rbcL
5' RNA, where processing to
The 5'-UTR and coding region are each necessary, but not in themselves
sufficient, for p47 binding to occur. p47 recognition could involve an
RNA secondary structure in one region that is formed or stabilized by
the presence of the other region. It is also possible that this binding
requires recognition and contact with distinct sequences present in
both regions. As shown in Fig. 1, protein-protected RNA fragments were
derived from coding as well as noncoding regions, indicating that one
or more proteins contact rbcL 5' RNA at multiple sites. A
requirement for both UTR and non-UTR sequences in plastid RNA-protein
binding and post-transcriptional gene regulation has been reported in
other studies (36, 50).
p47 binding to rbcL 5' RNA occurred in plastid extracts from
both L and DS plants and from leaves as well as cotyledons. Thus p47
binding activity was strictly correlated with polysome-association of
fully processed rbcL mRNAs and was not specific to a
developmental process associated with only one of these photosynthetic
organs. Since p47 binding to rbcL 5' RNA occurred in both mp
and bs plastid extracts, this protein cannot be directly implicated in
post-transcriptional regulatory processes that determine
C4-type cell-specific gene expression patterns
characteristic of mature amaranth leaves (12). p47 may have an
additional function(s) or interact with additional non-cell
type-specific mRNAs in this C4 plastid type.
Alternatively, although we have shown that bs/mp specificity is
maintained for several plastid mRNAs during the incubation times
required for protoplast purification and plastid purification (12), it
is possible that lower levels of p47 cross-linking observed in the mp
plastid extracts were due to some loss of bs specificity for the
(likely nuclear) genes encoding p47 during preparation.
Taken together, our analysis of light-associated p47 binding to 66); transcripts with longer 5'-UTRs did not associate
with p47 in vitro. Variations in the length of the
rbcL 5'-UTR were found to occur in vivo, and
these different 5' termini may prevent or enhance light-associated p47
binding, possibly affecting rbcL expression as well. p47
binding correlates with light-dependent rbcL
polysome association of the fully processed transcripts in photosynthetic leaves and cotyledons but not with cell-specific rbcL mRNA accumulation in bundle sheath and mesophyll chloroplasts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
s
1, or in a greenhouse bay equipped with
supplemental sodium vapor lamps to provide 14 h per day of
illumination (these two growth conditions gave identical binding
activities). Light-grown (L) soluble chloroplast protein extracts, or
total RNA, was prepared from fully expanded leaves of L plants, or for
some experiments cotyledons were harvested from L seedlings 6 days
after planting. To prepare etioplast (E) protein extracts or RNA,
cotyledons were harvested from seedlings germinated and grown in total
darkness for 6 days, as previously described (8). Dark-shifted (DS) plastid protein extracts or RNA were prepared from cotyledons of plants
that were grown under normal illumination for 6 days and then
transferred to darkness for 4 h before harvesting.
80 °C for 15 min. Protein-protected RNA fragments were pelleted through a 0.25 M sucrose cushion for 30 min at 70K in a TLA 100.3 rotor in
a Beckman TL 100 ultracentrifuge at 4 °C. The pelleted fragments
were treated with proteinase K for 30 min at 37 °C and then
extracted twice with phenol/chloroform. RNA fragments were precipitated
with ethanol, pelleted, dried, and resuspended in RNase-free water.
-32P]UTP in T7
transcription buffer (Epicentre Technologies), and 50 units of T7 RNA
polymerase for 1 h at 37 °C. After transcription, 1 unit of
RNase-free DNase was added to the reaction, and after 15 min of
incubation 1 µl of 0.5 M aurintricarboxylic acid was added. Labeled transcripts were purified on 5% acrylamide, 7 M urea gels. The labeled RNA bands were cut out of the gels
and crushed with a siliconized glass stirring rod. High salt buffer (0.5 M NaCl, 0.1 M Tris, pH 8.0, 20 mM EDTA) was added to the crushed gel slice, and the RNA
was allowed to elute for 10 min at room temperature. The eluted samples
were then phenol/chloroform/isoamyl alcohol-extracted and precipitated
with an equal volume of isopropyl alcohol. The resulting RNA
pellets were resuspended in RNase-free water and [32P]UTP
incorporation determined by liquid scintillation counting. Larger
amounts of unlabeled RNA for use in competition analyses were made in a
10-fold increased reaction volume and with the addition of 10 mM MgCl2 and 1 mM of all NTPs.
-mercaptoethanol, 12.5% glycerol, and 0.001% bromphenol blue were added to each sample. The
samples were boiled for 5 min, loaded onto 12.5% SDS-polyacrylamide gels, and separated at 30 mA for 3 h at room temperature. Gels were dried and analyzed using a PhosphorImager as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
66
(mapped by primer extension, data not shown) and the initiator AUG. For
L plants (lane L), one of the most significant protected
sites occurred adjacent to the 5' terminus, extending from
60 to
66
(lane L, band d). This protected fragment was clearly
present in RNAs from L seedlings, but was observed only weakly in DS
seedlings (lane DS), and not at all in E seedlings
(lane E). There were several additional nuclease-protected
fragments from further downstream in the 5'-UTR. One of these occurred
with L plants at an AU-rich region (lane L, band c) but not
with E or DS plants. Another protected fragment mapped to the
Shine-Dalgarno sequence (lanes L and DS, band b) and was observed in RNAs from L and DS plants but not with E
seedlings.
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Fig. 1.
Light-dependent protein-protected
fragments at the 5' end of rbcL mRNA. The
positions of RNA-binding proteins on polysome-associated
rbcL mRNAs were determined by RNA heelprinting analysis
as described under "Experimental Procedures." Arrows
indicate protein-protected fragments and the corresponding region in
the 5'-UTR region. E, 6-day-old etiolated seedlings.
DS, 6-day L seedlings transferred to darkness for 4 h.
L, 6-day-old light-grown seedlings. C,
single-stranded template DNA alone (control). The sequence to the
left represents the 5'-UTR of rbcL mRNA, from
the initiator AUG up to and including the 5'-processing site at 66.
The strong band near the top of the gel in all of the lanes
represents a stop site that is not dependent on the presence of
protein.
66, extending downstream through
the UTR, and containing an additional 60 nucleotides of protein coding region). Incubation of the labeled 5' RNA with L plastid extract resulted in a well defined mobility shift band on nondenaturing polyacrylamide gels (Fig. 2, 5'
LRP), which was not visible when this RNA was incubated with
purified E plastid extracts (data not shown). Formation of the 5' LRP
in L plastid extracts was protein-mediated, since it did not occur in
control reactions with no protein extract, with extracts that had been
boiled, or protease-treated (data not shown). Thus the GMS assays, like
the heelprinting studies, indicated the presence of light-associated rbcL 5' RNA binding protein activity. As shown in Fig. 2,
competition studies using unlabeled heterologous RNA of similar size to
the rbcL 5' RNA (7z-AS, a 130-nt RNA from a yeast RNA virus)
(45) indicated that formation of the 5' LRP complex in L extracts is specific. This complex could be competed with as little as 1× molar
excess of unlabeled self-competitor, whereas competition did not occur
with up to a 10,000-fold excess of the yeast viral RNA.
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Fig. 2.
Light-dependent
rbcL 5' RNA-protein complex formation is competed with
increasing molar concentrations of unlabeled self but not with a
heterologous RNA. Plastid extracts (ext.) from
light-grown plants (10 µg) were incubated with
32P-labeled rbcL 5' RNA (15 fmol) in the
presence of increasing molar concentrations of unlabeled
self-competitor or a yeast virus RNA (7z-AS). Reactions were
analyzed for RNA-protein complex formation by gel mobility shift assay
as described under "Experimental Procedures." 5'LRP,
shifted RNA-protein complex. 5'F, free unshifted RNA. The
uppermost band in the free 5' RNA lane (1st lane) represents
an additional secondary structure that this RNA adopts under
nondenaturing conditions.
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Fig. 3.
UV cross-linking of proteins to
rbcL 5' RNA is light-dependent and highly
specific. In vitro synthesized rbcL 5' RNA
was incubated with 20 µg of plastid extract as described for Fig. 3,
and the reactions were subjected to UV-cross-linking. The binding
reactions were then treated with RNase, boiled, and analyzed by
SDS-PAGE. A, plastid extracts (ext.) prepared
from light-grown or etiolated (etiol.) plants were incubated
with 32P-labeled rbcL 5' RNA in the absence ( )
or presence (+) of unlabeled self-competitor. B,
32P-labeled rbcL 5' RNA was incubated with
plastid extract from light-grown plants in the presence of the
appropriate competitor RNA, prior to UV cross-linking. Competitor RNAs
were unlabeled rbcL 5' RNA, 7z-AS, poly(A), and the
psbA 5' RNA, as indicated at the top of each
panel. The major doublet band of the light extracts at 47 kDa is
indicated.
59 to
69,
depending on the plant species. Both longer and shorter forms of
rbcL mRNAs have been found in association with polysomes
(3, 48), and differential processing at the rbcL 5' end has
been shown to affect LSU translation (40). These observations raise the
possibility that differential processing of the rbcL 5'-UTR
could affect the efficiency of its translation by altering RNA/protein
interactions within the UTR.
14 (which includes the Shine-Dalgarno sequence),
66 (corresponding
in size and sequence to the processed mature rbcL mRNA),
and
116 and
155 (composed of 116 and 155 nucleotides downstream of
the start AUG codon, respectively). An additional construct,
155A,
was similar to
155 but lacked the coding region and the
Shine-Dalgarno sequence.
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Fig. 4.
Light-dependent protein binding
to different length rbcL 5'-UTR RNAs and in
vivo differences in the rbcL 5'-UTR.
A, schematic representation of the 5' rbcL
mRNA fragments used for protein binding analysis. 14,
an RNA fragment that includes 14 nt upstream of the AUG translation
start site plus 60 nt of coding region.
66, an RNA
corresponding in size and sequence to the processed mature
rbcL mRNA, with a 66-nt long UTR upstream of the AUG
plus 60 nt of coding region.
116, RNA with 116 nt upstream
of the AUG plus 60-nt coding region.
155, RNA with
additional 155 nt upstream of the mature rbcL
mRNA-processing site plus 60 nt of coding region.
155A, same as above with the Shine-Dalgarno and coding
regions deleted. B, UV cross-linking of proteins to the
various length 5'-UTR probes. C, 5' primer extension
analysis of rbcL 5'-UTRs in vivo. Total mRNA
isolated from cotyledons of etiolated (E) or light-grown
(L) plants was hybridized with a 32P-labeled
primer corresponding to rbcL 5' coding sequence and extended
with reverse transcriptase. Extensions were separated on a
polyacrylamide urea gel, visualized, and quantified with a
PhosphorImager. 2.8 times more total RNA was used for the D reactions
than for the L reactions, to approximately equalize amounts of the
rbcL template. D, protein binding to
rbcL transcripts found in light-grown plants. B
and C, 32P-labeled RNA fragments corresponding
to different regions of the 5'-UTR of rbcL were incubated
with plastid extract in the absence (
) or presence (+) of unlabeled
self-competitor and UV cross-linked as described for Fig. 3.
66.
The longer
116 and
155 5'-UTR RNAs, both of which extended upstream and included all of the sequences found within the
66 RNA, showed no
cross-linking to p47. The
14 RNA also did not cross-link to p47 but
did show very strong cross-linking to one or more proteins of ~23
kDa. A cross-linked 23-kDa band of reduced intensity also occurred in
the
66 lane (which included all of the sequences in the
14 RNA plus
52 additional upstream nucleotides). It is possible that this same
smaller protein interacted with both RNA fragments, since it binds
downstream of the p47-binding site. The longer 5'-UTR fragments also
showed several less intense bands, some of which appeared to migrate at
the same position as some of the bands in
66 and
14 RNA lanes.
Interestingly, the
155A RNA, which lacked the Shine-Dalgarno and
coding sequences, did not show any cross-linking, whereas the longest
length UTR (
155) did cross-link to some proteins.
66,
corresponding to the 5' end of mature processed rbcL 5'
transcript, and the other at
174, corresponding in size to a typical
dicot rbcL primary transcript (47). An additional prominent
extension product was primarily in RNA isolated from L plants (Fig.
4C), where there was a clearly observable and abundant
extension product terminating at
103. In L RNAs the ratio of
transcripts terminating at
103 to
66 (determined by PhosphorImager
quantitation) was 1/3, indicating that a significant amount of
rbcL mRNA accumulating in vivo contained a
5'-UTR that extended beyond the
66 processing site but was still
significantly shorter than the primary transcript. In contrast to L
plants, the
103 rbcL transcripts were greatly reduced in the E plants (Fig. 4C), where only trace amounts of the
103 product were present (
103/
66 ratio of <1/20). These findings
indicate light-associated differences in the rate or mechanism of RNA
processing within the 5'-UTR of the amaranth rbcL
transcript, with a prominent intermediate transcript accumulating in
the light.
66
transcript, cross-linked to p47. rbcL RNAs corresponding to
the in vivo 5'-UTRs terminating at more upstream sites
(
81, a very minor extension product in light RNAs, and
103) showed
no p47 cross-linking. However, it is interesting to note that
transcript corresponding to the in vivo
103 UTR, the most
prominent intermediate 5' end in vivo, did show binding to a
different sized protein, of ~50 kDa.
14 and the
66 end of mature processed RNA. Furthermore, when additional 5'-UTR
sequences were present on the transcript, binding of p47 was
eliminated. The occurrence of longer rbcL 5'-UTRs in
vivo, together with the finding that extra upstream UTR sequences prevent p47 binding in vitro, suggests that complete
processing of the primary rbcL transcript to the
66
position may be necessary for this binding activity.
66) or only the coding region
(from +1 to +60). Fig. 5 shows that
neither of these regions (UTR or CR, respectively) alone could be
cross-linked to the p47 doublet. Only the 5' RNA probe (
66)
containing both regions showed this binding activity.
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Fig. 5.
The 5'-UTR and 5'-coding region are required
for p47 binding. 32P-Labeled RNA fragments consisted
of different regions of the rbcL 5' RNA probe, as diagrammed
to the left of the gel panel. 66, the entire 5'
RNA region, from
66 to +60. UTR, only the UTR portion of
the rbcL 5' RNA from +1 to
66. CR, only the
coding region from +1 to +60. These were incubated with plastid extract
and UV cross-linked as described for Fig. 3.
66, it might be expected that differences in 5'-UTR
length would be associated with differences in polysome association.
Fig. 6A shows a sucrose
gradient profile of polysomes obtained from L cotyledons. The
distributions of rbcL mRNAs within different regions of
the polysome gradient were similar to that reported for L seedlings in
previous studies (8, 9). Primer extensions from these L
polysome-associated rbcL mRNAs (Fig. 6B) indicated that there were no differences in distribution for
transcripts terminating at the various 5' locations. mRNAs ending
at
66,
103, and the
174 primary transcripts all showed similar
patterns of distribution along the polysome (fractions 2-5) and
monosome (fractions 6 and 7) regions of the sucrose gradient.
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Fig. 6.
Polysome association of in vivo
processed rbcL transcripts. A,
sucrose gradient profile of polysome preparations from cotyledons of
light-grown amaranth seedlings. Crude polysome extract was loaded onto
the gradient; sedimentation is from right to
left. B, 5' primer extension analysis of
rbcL mRNA populations associated with polysome
(P, fractions 2-5) and monosome (M,
fractions 6 and 7) regions of the sucrose
gradient. RNA was isolated from each fraction by phenol extraction and
ethanol precipitation and extended using the rbcL 5' primer
and extension conditions described for Fig. 4C.
131 and
155. Thus, whereas p47-kDa protein binding activity was observed only with the
66 mature transcripts in L plastid extracts, mRNAs with longer 5'-UTRs that are not capable of binding to this protein in
vitro still appear capable of associating with polysomes of L
seedlings in vivo.
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Fig. 7.
Binding to the 5'-UTR of
rbcL mRNA is light-regulated in photosynthetic
tissues and is not highly cell type-specific. Protein extracts
were prepared from plastid isolated plants grown under various
illumination conditions or from various tissues and cell types, or from
purified and separated bundle sheath and mesophyll chloroplasts, as
indicated at the top of the figure. Light,
light-grown mature leaves. Etiolated, 6-day-old dark-grown
cotyledons. Bundle Sheath, separated and purified
chloroplasts from leaf bundle sheath cells. Mesophyll,
separated and purified chloroplasts from leaf mesophyll cells. 6 day dark-shift cots, cotyledons grown in light for 6 days and then
shifted into complete darkness for 4 h. 6 day light
cots, cotyledons from plants grown under standard illumination for
6 days. Each lane represents a standard cross-linking reaction,
incubated in the absence ( ) or presence (+) of excess
self-competitor. The position of the 47-kDa doublet is indicated.
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Fig. 8.
Comparison of protein cross-linking patterns
of two plastid-encoded mRNAs. Plastid protein extracts were
incubated and UV cross-linked with in vitro synthesized and
labeled transcripts as described for Fig. 3, using fragments
corresponding to the 5' regions of two plastid RNAs. rbcL,
the 66 5' RNA. psbA, 133 nt 5' fragment of the amaranth
psbA transcript containing 87 nt of UTR sequence and 46 nt
of coding sequence. psbA lane 1, binding in the absence of
any added plastid RNA competitor. psbA lane 2, competition
with 10,000× molar excess of unlabeled psbA RNA. psbA lane
3, competition with 10,000× molar excess of unlabeled rbcL
RNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21. Light-dependent binding to an
AU-rich region within the 5'-UTR of psbA mRNA has been
observed in other systems (23, 29). RNase protection at the 5' end of
processed chloroplast mRNAs, similar to that shown here for
rbcL, has been reported for the ATPase synthase gene cluster
of spinach (51). It is possible that binding of proteins at or near the
5' terminus of some plastid transcripts is required for their
translation or stabilization in the light.
14
(relative to the initiator AUG) and the end of the processed
rbcL 5'-UTR at
66. The addition of as few as 14 nucleotides corresponding to sequences upstream of the normal
5'-processing site completely eliminated detectable p47 binding in our
in vitro assay. Secondary structures within the 5'-UTRs of
prokaryotic and eukaryotic mRNAs can affect the binding of
regulatory factors and affect their translation (16, 40, 55-57), and a
predicted secondary structure occurring within the
rbcL-66 5'-UTR is a potential target for p47
binding. The additional 5' sequences could prevent binding of the
47-kDa binding proteins by interfering with normal secondary structure
conformations. Alternatively, the extra 5' rbcL sequences
could play a more active role in preventing binding of the 47-kDa
proteins through direct interactions with additional regulatory
factors. Several unique cross-linked bands were observed with RNAs
containing sequences upstream of
66 (Fig. 4B). Proteins binding upstream of
66 could work by modulating structural changes in
the p47 target sequence or by directly overlapping and blocking access
to the target sequence.
66 is necessary for p47 binding,
processing of the psbA 5'-UTR did not affect binding of a
specific mRNA-binding complex.
66
rbcL 5' RNA suggests three possible functions for this protein. First, p47 could be responsible for translation-associated processing of rbcL mRNA to the
66 position. Immediate
polysome association of the full-length transcript in L seedlings could be accompanied by cotranslational processing during protein synthesis, so that intermediate length transcripts are present only on polysomes in the light. Second, p47 could be responsible for capping and protecting the polysome-bound rbcL mRNAs from
degradation following cleavage at the
66 site. 5'-UTRs of plastid
mRNAs have been implicated in control of transcript stability in
other systems (26, 36, 37, 50, 58). Third, p47 could be one of a small
group of regulatory proteins that specifically bind to different length rbcL 5'-UTRs that accumulate in vivo (in this
case the
66 mRNA), mobilizing these transcripts to polysomes. If
this were the case, then other proteins, such as the 50-kDa protein
that binds to the
103 rbcL RNA, could selectively activate
the other rbcL transcripts. These possible functions are not
exclusive; p47 could potentially have multiple roles in association
with polysomes that are translating the rbcL mRNAs.
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ACKNOWLEDGEMENTS |
---|
We thank Stephen Mayfield, Jeremy Bruenn, Margaret Hollingsworth, Joseph Boinski, and Amy Corey for helpful advice and discussions and Jim Stamos for assistance in preparing the illustrations.
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FOOTNOTES |
---|
* This work was supported by National Science Foundation Grants MCB 9316806 (to J. O. B.) and MCB 9728547 (to J. O. B. and P. D. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Imaging Research, Inc., Brock University, St.
Catherines, ON, CA.
§ To whom correspondence should be addressed. Tel.: 716-645-3488; Fax: 716-645-2975; E-mail: camjob@acsu.buffalo.edu.
Published, JBC Papers in Press, November, 13, 2000, DOI 10.1074/jbc.M009236200
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ABBREVIATIONS |
---|
The abbreviations used are: RuBPCase, ribulose 1,5-bisphosphate carboxylase; LSU, large subunit of RuBPCase; GMS, gel mobility shift assay; LRP, large RNA mobility shift complex; p47, 47-kDa rbcL 5' RNA binding protein; L, light-grown plants; E, etiolated plants; DS, dark-shifted plants; bs, bundle sheath cells; mp, mesophyll cells; UTR, untranslated region; nt, nucleotide; 7z-AS, antisense, 3'-UTR from yeast double-stranded RNA virus.
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