Light-associated and Processing-dependent Protein Binding to 5' Regions of rbcL mRNA in the Chloroplasts of a C4 Plant*

Dennis J. McCormacDagger, Hanz Litz, Jianxin Wang, Paul D. Gollnick, and James O. Berry§

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-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.

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 -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.

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 [alpha -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.

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 beta -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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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.

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 -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.

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.



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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.

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 -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.

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 -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.

As shown in Fig. 4B, the predominant doublet band at 47 kDa was observed only with the rbcL 5' RNA terminating at -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.

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 -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.

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 -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.

Fig. 4 indicates that in L plastid extracts, sequences required for p47 binding occur within a 52-nt region of the 5'-UTR, between -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.

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 -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.

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 -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.

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 -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.

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).



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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.

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.



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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

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 -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.

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 -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.

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 -66 is necessary for p47 binding, processing of the psbA 5'-UTR did not affect binding of a specific mRNA-binding complex.

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 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.


    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.


    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.

Dagger 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


    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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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES


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