Processing of the psbA 5′ Untranslated Region in Chlamydomonas reinhardtii Depends upon Factors Mediating Ribosome Association

Unless otherwise noted, all C. reinhardtii strains were grown at 25°C under constant light in complete media (Tris-acetate-phosphate; Harris, 1989) to a density of ∼10⁶ cells/ml. Cells were harvested by centrifugation at 4°C for 5 min at 4,000 g, washed with H₂O, and pelleted again at 4,000 g for 5 min at 4°C. Cell pellets were frozen in liquid N₂ and stored at −70°C.

Total and polyribosome-associated RNAs were prepared according to published protocols (Barkan, 1988; Cohen et al., 1998). To prepare membrane-associated RNA, the ground frozen cell pellet was twice extracted with extraction buffer lacking the detergents Triton X-100 and polyoxyethelene-10-tridecyl-ether to remove soluble RNA. The insoluble pellet was then resuspended in extraction buffer containing these detergents and used to prepare polyribosome-associated RNA.

Approximately 1 pmol of 5′-radiolabeled DNA oligonucleotide complementary to positions +47 to +32 of the psbA coding region (5′-CGAGCCCATAGGCTAG-3′) or positions +66 to +49 of the psbD coding region (5′-AAGCCAGTCATCAGCGTC-3′) was annealed to 5–25 μg RNA in a 7-μl mixture containing 50 mM Tris (pH 8.3), 60 mM NaCl, and 10 mM DTT by slow cooling from 80°C to room temperature. The reaction mixture was incubated at 42°C for 30 min after addition of 8 μl reaction mixture (50 mM Tris, pH 8.3, 60 mM NaCl, 10 mM DTT, 15 mM MgCl₂, 1.25 mM each dNTP, and 0.2 U AMV reverse transcriptase; Life Sciences Inc., St. Petersburg, FL). The RNA was degraded by addition of 20 μl degradation buffer (50 mM Tris, pH 7.5, 0.1% SDS, and 7.5 mM EDTA, pH 8.0) and 3.5 μl 3 M KOH followed by incubation at 90°C for 3 min then 42°C for 25 min. The sample was neutralized upon addition of 6 μl acetic acid and 10 μl 3 M NaOAc (pH 5.3), EtOH precipitated, and resuspended in gel loading buffer. Products were separated by electrophoresis in a 7.5% polyacrylamide/8 M urea gel and quantified by PhosphorImager analysis. Samples were loaded to achieve approximately equal signals and do not reflect the relative amounts of psbA mRNA in each strain of C. reinhardtii.

100 pmol of a DNA oligonucleotide complementary to positions +171 to +156 of the psbA coding region (5′-TGGCGGAGCAGCGATG-3′) was annealed to 5 μg wild-type total RNA or 15 μg hf261 total RNA in a 3.5-μl mixture containing 0.5 μl buffer (200 mM Tris, pH 7.5, 200 mM KCl, 1 mM EDTA, pH 8.0, and 1 mM DTT) and 0.1 U PRIME RNase inhibitor (5 Prime → 3 Prime, Inc., Boulder, CO) by slow cooling from 80°C to room temperature. RNA cleavage was initiated upon addition of 0.5 μl 100 mM MgCl₂, 0.5 μl 0.2 U/μl PRIME RNase inhibitor, and 0.5 μl RNase H (2.2 U/μl; GIBCO BRL, Gaithersburg, MD). Reactions were incubated at 37°C for 30 min. Products were separated by electrophoresis in a 5% polyacrylamide/8 M urea gel. RNA blotting and hybridization with a random-primed, [³²P]-labeled psbA 5′ cDNA fragment (HindIII-ClaI) were performed as described previously (Mayfield et al., 1994).

Approximately 1 μg total protein from C. reinhardtii, partially purified by heparin-agarose chromatography according to Cohen et al. (1998), was incubated for 10 min at room temperature with 0.5 U PRIME RNase inhibitor in a total volume of 8 μl dialysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM KOAc, 0.2 mM EDTA, pH 8.0, 2 mM DTT, 20% glycerol) supplemented with 4 mM MgCl₂. To each sample, 0.08 pmol of in vitro transcribed [³²P]-labeled RNA (beginning with two G residues added 90 bases [−90] or 36 bases [−36] upstream of the start codon and ending 171 bases downstream of the start codon), 20 μg of wheat germ tRNA (Sigma), and 3 μg of FuD7 (a C. reinhardtii strain lacking the psbA gene) total RNA were added and the reaction was incubated at room temperature for 10 min. Where indicated, 8 pmol unlabeled in vitro transcribed RNA was also added as a competitor. RNA–protein complexes were separated in a 5% nondenaturing polyacrylamide gel.

UV cross-linking assays were performed as gel mobility shift assays with the following alterations. Approximately 5 μg heparin-agarose purified protein was incubated with 1.0 pmol of internally labeled RNA (−90 or −36) generated by in vitro transcription using both [α-³²P]ATP and [α-³²P]UTP. After complex formation, the reactions were irradiated with short-wave UV light for 1 h at 4°C. The RNA was digested at 55°C for 45 min after addition of urea, EDTA (pH 8), and RNase A to final concentrations of 3 M, 3.75 mM, and 0.5 mg/ml, respectively. Labeled proteins were separated by SDS-PAGE and visualized using a PhosphorImager.

RAC was performed as described by Cohen et al. (1998). In brief, 125 μg of in vitro transcribed RNA (−90 or −36) was coupled to 100 μl amino gel 1702 (Sterogene Bioseparations Inc., Carlsbad, CA). 1 ml heparin-agarose purified protein was passed over the resulting columns, washed, and recovered by elution with a high salt buffer. Equal quantities of RAC proteins were mixed with 2× sample buffer (5% SDS, 5% β-mercaptoethanol, 400 mM Tris, pH 6.8, 10% sucrose), heated to 65°C for 5 min, and separated by SDS-PAGE. The gels were either stained with Coomassie blue or electroblotted to nitrocellulose (Schleicher and Schuell, Keene, NH) in 10 mM CAPS (pH 11)/10% methanol (Mayfield et al., 1994). Filters were treated with rabbit polyclonal antisera specific for RB38, RB47, or RB60.

S1 mapping of the psbA mRNA from C. reinhardtii previously identified two 5′ termini, one 90 nt upstream of the start codon and a more pronounced terminus 36 nt from the start codon (Erickson et al., 1984). Multiple 5′ termini in this assay may reflect multiple transcription start sites, posttranscriptional cleavage events, or RNA modifications or structural elements which result in poor hybridization of the radiolabeled probe. We used primer extension analysis to examine the 5′ termini of the psbA 5′ UTR. A radiolabeled oligonucleotide complementary to the 5′ coding region of psbA was annealed with total RNA prepared from wild-type C. reinhardtii. Reverse transcriptase was used to extend the probe to the 5′ terminus of the message. Extension products were separated by PAGE and quantified by PhosphorImager analysis. As shown in Fig. 1, 95% of the psbA message contains a 5′ terminus 36 nt upstream of the start codon whereas the remaining psbA mRNAs feature 5′ UTRs of 90 nt in length. Similar results were observed previously by primer extension analysis (Shapira et al., 1997).

The two predominant extension products could reflect different 5′ termini or may indicate partial blocks in primer extension resulting from strong structural elements within the 5′ UTR. To resolve this issue we used RNase H to cleave the psbA mRNA at the site of hybridization to a complementary oligonucleotide to generate 5′ psbA fragments of a size that could be resolved by PAGE and subsequently visualized by Northern analysis. The size of the wild-type psbA 5′ UTR can be compared with the 5′ UTR from the hf261 strain, a nuclear mutant in which only the 90-nt psbA 5′ UTR was observed by primer extension analysis (see Fig. 6). As shown in Fig. 2, the 5′ fragment of the psbA message from hf261 is longer than the predominant wild-type psbA 5′ fragment, confirming that the primer extension products reflect 5′ UTRs of differing size. Therefore, the vast majority of wild-type psbA transcripts from C. reinhardtii begin 36 nt upstream of the start codon, adjacent to the RBS (Fig. 3 A).

Neither primer extension analysis nor Northern analysis can distinguish between 5′ termini generated by transcription initiation and termini generated by posttranscriptional mRNA processing. However, the data will show that the appearance of the 36-nt 5′ UTR is most closely associated with posttranscriptional events, suggesting that this terminus most likely results from mRNA processing. This conclusion is further supported by the observation that partial deletion of a putative promoter element required for transcription initiation of the of the −36 terminus (Erickson et al., 1984) had no affect on the appearance of the 36-nt 5′ UTR (Loop-del [Mayfield et al., 1994]; data not shown).

To identify RNA elements required for processing, site-specific mutations were engineered within the 5′ UTR of the psbA gene which was then reintroduced into the C. reinhardtii chloroplast genome by particle bombardment (Mayfield et al., 1994). One of these constructs, Alter, replaced the four uridine nucleotides just upstream of the RBS with adenines (Fig. 3,B). In addition to disrupting the stem-loop element, this mutation changed the primary sequence at the processing site. This mutation results in a 95% reduction in D1 protein synthesis and an 80% reduction in D1 protein accumulation (Mayfield et al., 1994). However, primer extension analysis of total RNA isolated from this strain demonstrates that cleavage of the psbA 5′ UTR is identical in extent and location to processing in wild-type C. reinhardtii (Fig. 4), indicating that processing is not a function of the primary RNA sequence at the cleavage site.

Many chloroplast-encoded mRNAs have a sequence resembling a prokaryotic-like SD that serves as an RBS. However, because these sequences tend to vary in size and location relative to their Escherichia coli counterparts, the relevance of these RBSs in chloroplasts has been questioned (Fargo et al., 1998). Deletion of a putative SD sequence in the 5′ UTR of the C. reinhardtii psbA mRNA (RBS-del) eliminated psbA mRNA polyribosome association (Yohn et al., 1996) and D1 synthesis in vivo (Mayfield et al., 1994), suggesting that this sequence is a functional RBS. Additional mutations designed to disrupt the RBS have been introduced into the psbA 5′ UTR (Fig. 3,B). These mutations either eliminated the RBS (RBS-del, RBS-Alt), sequestered the RBS in an extended stem-loop structure (RBS-paired), or changed the location of the RBS by replacing the wild-type RBS with another RBS placed upstream of the stem-loop element (RBS-upstream). All of these changes block ribosome association and abolish D1 protein synthesis in vivo, providing further evidence that this SD sequence functions as an authentic RBS (Mayfield et al., 1994; Bruick, R.K., and S.P. Mayfield, unpublished results). Total RNA was prepared from each of these mutants and primer extension analysis was used to characterize the processing of the psbA 5′ UTR. As shown in Fig. 5, each of these mutants possess only one 5′ terminus corresponding to the unprocessed psbA 5′ UTR. These data show that a functional RBS, required for ribosome association and psbA translation, is also required for normal processing of the 5′ UTR.

Though processing of the psbA 5′ UTR requires a SD sequence, and presumably initial ribosome association, processing is not dependent upon D1 protein synthesis. For example, a deletion within the psbA 5′ UTR that positions the RBS 11 nt upstream of the start codon (RBS-11, Fig. 3,B) reduces psbA association with large polyribosomes and eliminates D1 protein synthesis in C. reinhardtii while enhancing translation of the same message in E. coli (Bruick, R.K., and S.P. Mayfield, manuscript in preparation). As demonstrated by a primer extension assay, the psbA 5′ UTR from this mutant is still cleaved to the same extent and in the same position relative to the RBS as in the wild type (Fig. 5). The exact location of the processing site was confirmed by RNA sequencing (data not shown). Thus, we imagine that the RBS in this construct remains accessible for recognition by factors involved in the early assembly of a translation initiation complex before processing but that this mutant RBS lacks the ability to specify the correct initiation codon.

Several nuclear mutations have been characterized in C. reinhardtii that specifically affect expression of the D1 protein (Yohn et al., 1996; Yohn et al., 1998b). In each of these mutants (F35, hf149, hf233, hf261, hf859, and hf1085), D1 protein synthesis is specifically lacking. While the primary defects of these mutations are not known, psbA association with polyribosomes is reduced or absent in the majority of these strains, suggesting that these mutations affect translation initiation (Yohn et al., 1996; Yohn et al., 1998b). Total RNA was prepared from each of these mutants and primer extension analysis of the psbA 5′ UTR was performed (Fig. 6). Each of these nuclear mutations has a pronounced effect on the amount, but not the position, of processing of the psbA 5′ UTR. In each case, the ratio of transcripts featuring a 90-nt 5′ UTR (−90) to processed transcripts (−36) is increased compared with the wild type, with some mutants lacking any detectable psbA terminus 36 nt upstream of the initiation (hf261, hf1085).

Just as these nuclear mutants specifically affect translation of the psbA mRNA, the effect on mRNA processing also appears to be psbA specific. In addition to the psbA 5′ UTR, the 5′ UTR of the psbD mRNA (encoding the D2 protein) is also processed in C. reinhardtii (Rochaix et al., 1984). Primer extension analysis using a radiolabeled oligonucleotide complementary to the psbD 5′ coding region was performed on total RNA prepared from the wild type and the hf261 strain (Fig. 6). Whereas processing of the psbA 5′ UTR is completely absent in hf261, processing of the psbD 5′ UTR remains unaffected in this mutant as does D2 protein synthesis (Yohn et al., 1998b).

To determine if the psbA 5′ UTR is differentially processed in response to environmental conditions, total RNA was isolated from C. reinhardtii grown under continuous light or continuous darkness. Primer extension analysis indicated no difference in the relative amounts of the 5′ termini of the psbA mRNA in response to growth under continuous light or in constant darkness (Fig. 7). A previous study reported no change in psbA processing throughout the 18 h after a shift from low light to high light growth conditions (Shapira et al., 1997). These results suggest that cleavage of the psbA 5′ UTR is not directly related to light-dependent translational regulation.

During growth under constant light, only 35% of psbA mRNA is associated with large polyribosomes (Yohn et al., 1996). Polyribosome-associated RNA was isolated and processing of the psbA 5′ was analyzed by primer extension analysis. The extension products of psbA mRNA associated with polyribosomes were indistinguishable from those observed in total RNA (Fig. 7). Because the D1 protein is inserted into the membrane during, or soon after, synthesis, we examined the subset of polyribosome-associated psbA mRNA associated with thylakoid membranes. If this subset of mRNAs comprises the pool of functionally relevant psbA message, we might observe a difference in the amount of processed psbA 5′ UTR in this fraction as compared with total mRNA. Again, primer extension analysis indicated no difference in the relative amount of processed psbA 5′ UTR between membrane-associated mRNA and other RNA populations (Fig. 7).

The stem-loop element upstream of the RBS had been identified previously as a binding site for a protein complex involved in the dynamic regulation of psbA translation in response to light (Danon and Mayfield, 1991). However, as a consequence of processing, >95% of the steady-state levels of psbA message lacks this stem-loop element in vivo. To determine if the processed psbA 5′ UTR was still capable of being recognized by the protein complex, a gel mobility shift experiment was used to compare complex binding to the two psbA 5′ UTRs in vitro. Heparin-agarose purified proteins from wild-type C. reinhardtii were incubated with in vitro transcribed RNAs corresponding to either the 90-nt psbA 5′ UTR (−90) or the processed 5′ UTR (−36). Complex binding is reflected by the appearance of an RNA–protein complex with reduced mobility in a nondenaturing polyacrylamide gel. As shown in Fig. 8, both RNAs are bound by protein. Furthermore, each RNA is able to compete the binding of the complex to the other RNA when added in excess as a cold competitor of the labeled RNA for the protein complex. The possibility of the −90 RNA being processed to the −36 RNA in vitro before complex formation is excluded by the observation that the migration of the −90 RNA–protein complex is retarded relative to the −36 RNA–protein complex.

Previous experiments have identified the primary psbA binding protein to be RB47, the cPABP (Danon and Mayfield, 1991; Yohn et al., 1998a). UV cross-linking of both radiolabeled transcripts (−90 or −36) complexed with heparin-agarose purified proteins resulted in the specific labeling of RB47 (Fig. 9). The lower intensity of RB47 labeling using the −36 transcript may be due to fewer radiolabeled residues in close proximity to the bound cPABP for the shorter RNA fragment or may reflect slightly lower binding affinity of the 36-nt 5′ UTR compared with the 90-nt 5′ UTR.

RAC was used to further characterize complex binding to the processed 5′ UTR. RNA transcripts corresponding to the entire (−90) and processed (−36) psbA 5′ UTRs were immobilized on solid supports. Heparin-agarose– purified C. reinhardtii lysate was passed over each support and bound proteins were eluted with high salt. An identical set of proteins was eluted from both columns as observed by Coomassie blue staining (Fig. 10,A). Western analysis with antibodies specific for the cPABP (RB47), the chloroplast-localized protein disulfide isomerase (RB60) (Kim and Mayfield, 1998), and a 38-kD component of the psbA binding complex (RB38) further demonstrated that both psbA 5′ UTRs are recognized by an identical protein complex in vitro (Fig. 10 B). Taken together, these results predict that removal of the stem-loop element from the psbA 5′ UTR does not prevent the binding of the psbA-specific protein complex nor does it preclude the dynamic regulation of translation in response to light.

In both C. reinhardtii and higher plants, the expression of nuclear and chloroplast genes is tightly regulated to allow for a rapid response to fluctuations in environmental conditions. The regulation of chloroplast-encoded genes is dependent upon sequences within their 5′ UTRs that influence message stability, translation, and possibly message localization (Gillham et al., 1994; Mayfield et al., 1995; Rochaix, 1996). Mutational analysis of the 5′ UTR of the psbA mRNA has shown that RNA elements, including a consensus SD sequence and an upstream stem-loop element, mediate D1 expression (Mayfield et al., 1994). In this study, primer extension analysis of the psbA mRNA from a variety of C. reinhardtii mutants has revealed a correlation between processing of the 5′ UTR and association of the mRNA with ribosomes. Alterations to the psbA 5′ UTR that disrupt the RBS prevent RNA processing. Nuclear mutations that specifically block D1 protein synthesis, in part by reducing ribosome association, specifically reduce psbA mRNA processing. The proximity of the processing site to an accessible and competent RBS, and the influence on processing of nuclear-encoded factors that affect ribosome association, suggest a relationship between ribosome association and processing of the 5′ UTR of the psbA mRNA.

In contrast to psbA messages from C. reinhardtii, generally only one 5′ terminus, corresponding to the unprocessed 5′ UTR from C. reinhardtii, has been observed for the psbA mRNA isolated from higher plants (Zurawski et al., 1982; Link and Langridge, 1984; Sugita and Sugiura, 1984; Boyer and Mullet, 1986; Boyer and Mullet, 1988; Hanley-Bowdoin and Chua, 1988; Liere et al., 1995). Nevertheless, processing of mRNAs is a common phenomenon in higher plant chloroplasts, in which most messages are transcribed as polycistronic transcripts. For example, the petD mRNA, which is transcribed as a dicistronic message downstream of petA, contains elements within the 5′ UTR that are required for accumulation of the monocistronic petD mRNA. Failure to process the petD 5′ UTR results in the loss of translation of the petD message (Sakamoto et al., 1993; Sakamoto et al., 1994a,b).

Processing of the 5′ UTR of the rbcL transcript, the chloroplast mRNA encoding for the large subunit of Rubisco, is also correlated with ribosome association and protein synthesis. In C. reinhardtii, for example, changes in large subunit synthesis in response to light levels coincide with changes in processing of the 5′ UTR (Shapira et al., 1997). rbcL transcripts with longer 5′ UTRs (beginning 168 nt upstream of the start codon rather than 93 nt) are not associated with polyribosomes and probably are not translationally competent. Recovery of large subunit synthesis is accompanied by an increase in the relative level of processed mRNAs. In mature barley leaves, inhibition of large subunit protein synthesis by methyl jasmonate is accompanied by a change in the predominant rbcL 5′ UTR, resulting from alternative processing of the primary transcript. Once again, the longer 5′ UTR is not associated with polyribosomes (Reinbothe et al., 1993). These observations further suggest that in both C. reinhardtii and higher plants, processing of 5′ UTRs coincides with ribosome association of those mRNAs. Thus, although it is often assumed that 5′ end formation occurs before, and is necessary for proper translation initiation, the available data also support the possibility that processing of mono- and polycistronic 5′ UTRs may be a consequence of ribosome association, rather than a prerequisite for it.

Although processing of the C. reinhardtii psbA 5′ UTR appears to be linked with translation initiation at the level of small ribosomal subunit recognition of the SD sequence association, no correlation was observed between processing and the dynamic regulation of translation in response to illumination. When C. reinhardtii is grown in the dark rather than the light, D1 protein synthesis is reduced in conjunction with reduced binding activity of the cPABP complex. Nevertheless, no difference in processing was observed between psbA mRNA isolated from C. reinhardtii grown in the light or in the dark, nor is there a direct linear relationship between the amounts of light-regulated translational activator proteins, such as the cPABP, and processing in the nuclear mutants. Therefore, processing is dependent upon at least the initial stages of ribosome assembly at the SD sequence, which occurs before, and independent of, dynamic light-dependent translation. However, some proteins may be common to both of these processes.

Previous studies have demonstrated that mutations introduced within the psbA 5′ UTR upstream of the RBS can disrupt D1 expression. Typically, these mutants accumulate normal amounts of psbA mRNA but synthesize D1 protein at <5% of the wild-type level (Mayfield et al., 1994). Nevertheless, the psbA 5′ UTR in each of these mutants is processed normally. Processing within these transcripts removes the mutated RNA upstream of the RBS and results in a steady-state pool of psbA transcripts that is virtually indistinguishable from the wild type. Therefore, sequences upstream of the RBS must influence D1 expression before processing and ribosome association, possibly by acting on steps such as formation of a preinitiation complex or perhaps even in localization of the psbA mRNA to specific regions of the chloroplast (Rochaix, 1996). The D1 protein must be synthesized in close proximity to the thylakoid membrane, and translational activators, including the cPABP, have been shown to be associated with a specific membrane fraction within the chloroplast (Zerges and Rochaix, 1998). Perhaps these upstream sequences act in concert with translational activator proteins to target the psbA mRNA to membrane-associated ribosomes.

Sequences upstream of the processing site could also influence chloroplast gene expression at the level of message stability, acting before the early stages of ribosome association. For example, the 5′ UTR of the chloroplast psbD mRNA from C. reinhardtii is processed in a manner similar to that of the psbA mRNA (Rochaix et al., 1984; Erickson et al., 1986; Nickelsen et al., 1994). Analysis of nuclear mutants that destabilize the psbD mRNA has indicated that the sequences upstream of the processing site in the psbD 5′ UTR may mediate this mRNA stability by interacting with nuclear encoded factors (Nickelsen et al., 1994). Furthermore, a deletion engineered within the psbD 5′ UTR designed to initiate psbD transcription at the processing site resulted in a lack of psbD mRNA accumulation despite a wild-type rate of transcription of the mRNA (Rochaix, 1996). An analogous chloroplast deletion engineered to initiate psbA transcription at the processing site also resulted in failure to accumulate any psbA mRNA (data not shown). These data show that sequences upstream of the RBS can influence message stability, likely acting during the early stages of mRNA maturation. Interestingly, an endonucleolytic cleavage site associated with mRNA degradation has been mapped within the spinach psbA 5′ UTR in vitro. Similarities exist between this endonucleolytic cleavage site and the processing site within the C. reinhardtii psbA 5′ UTR. Both cleavage sites are located immediately upstream of a consensus RBS. In spinach, this site also defines the upstream boundary of the core RNA element recognized by an RB complex that includes the ribosomal protein S1 (Klaff, 1995; Alexander et al., 1998). The close proximity of this cleavage site in spinach psbA mRNA to the putative RBS and the site of ribosome assembly may reflect an intimate relationship between translation initiation, mRNA processing, and message stability. This relationship is also observed for psbA mutants in C. reinhardtii (RBS-del, RBS-Alt, RBS-paired) in which the loss of ribosome binding due to the absence of a SD sequence results in the absence of processing and a reduction in psbA mRNA accumulation (Mayfield et al., 1994).

Originally, mutations to the upstream stem-loop element of the psbA mRNA were thought to influence D1 translation by altering the binding site of the light-regulated cPABP complex as the stem-loop element was found to be protected by protein binding in a T1-gel mobility shift assay (Danon and Mayfield, 1991; Mayfield et al., 1994). However, gel shift assays, UV cross-linking, and RAC assays presented here all indicate that both the 90-nt and processed psbA 5′ UTRs are recognized by an identical set of psbA-specific RB proteins. Thus, dynamic light-regulated translation via binding of these activator proteins should still be possible with the truncated psbA mRNA.

Based on these and other results, we propose a model for psbA maturation and translation in the C. reinhardtii chloroplast. The psbA mRNA is transcribed with a 5′ UTR extending at least 90 nt upstream of the start codon. The 5′ UTR is rapidly processed to the −36 transcript but not before the upstream RNA elements influence D1 expression. These upstream elements could affect expression at a number of key steps including RNA stability, formation of a preinitiation complex, or mRNA localization. Processing is mediated by proteins, including ribosomal subunits and possibly other initiation factors, that recognize the RBS during the early stages of ribosome association. The nuclease responsible for processing has not yet been identified but appears to be closely associated with a ribosomal subunit or with a protein complex required for ribosome association. Interaction of the ribosomal subunits and associated proteins with the processed 5′ UTR may act to stabilize the message. Processing may also initiate an mRNA degradation pathway affecting the half-life of the transcripts. Whereas removal of the upstream sequences may be necessary to make the message competent for high levels of translation, processing alone is not sufficient for translation of the message as evidenced by mutations, both chloroplast and nuclear, that affect psbA translation without eliminating processing. Upon illumination, the binding activity of a set of psbA-specific RB proteins, including the cPABP, is enhanced in response to increased photosynthetic activity. Binding of this protein complex stimulates translational activity, probably acting via recognition of the psbA 5′ UTR through a stretch of adenine residues between the RBS and the start codon (Bruick, R.K., and S.P. Mayfield, manuscript in preparation). While the molecular basis underlying each of these events remains unknown, we have begun to define distinct RNA elements within the psbA 5′ UTR that are recognized by trans-acting factors to orchestrate message stability, mRNA processing, general translational competency, and dynamic translational activation.

We thank Christopher Yohn and Amybeth Cohen for critical reading of this manuscript and for providing membrane-associated RNA (A. Cohen).

This work was supported by funds from the National Institutes of Health to S.P. Mayfield (GM54659). R.K. Bruick was supported by a National Science Foundation (NSF) predoctoral fellowship and an NSF Graduate Research Traineeship in Macromolecular Structure and Plant Biology.

cPABP

chloroplast poly(A)-binding protein

nt

nucleotide

RAC

RNA affinity chromatography

RB

RNA-binding

RBS

ribosome binding sequence

SD

Shine-Dalgarno

UTR

untranslated region