Translation of the Chloroplast <i>psbA</i> mRNA Requires the Nuclear-encoded Poly(A)-binding Protein, RB47

Yohn, Christopher B.; Cohen, Amybeth; Rosch, Cristen L.; Kuchka, Michael R.; Mayfield, Stephen P.

doi:10.1083/jcb.142.2.435

Cited by 73 publications

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“…The Plant Cell Nuclear genes in Chlamydomonas that are required for the translation of the chloroplast psbA (Girard-Bascou et al, 1992;Yohn et al, 1998b), psbC (Rochaix et al, 1989;Zerges and Rochaix, 1994), psaB (Stampacchia et al, 1997), and atpA (Drapier et al, 1992) mRNAs have been identified by genetic analysis. A mutation in each of these genes disrupts the translation of a specific chloroplast mRNA, suggesting that each mRNA requires specific translational activators.…”

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confidence: 99%

A Nuclear Gene in Maize Required for the Translation of the Chloroplast atpB/E mRNA

McCormac¹,

Barkan²

1999

The Plant Cell

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To elucidate mechanisms that regulate chloroplast translation in land plants, we sought nuclear mutations in maize that disrupt the translation of subsets of chloroplast mRNAs. Evidence is presented for a nuclear gene whose function is required for the translation of the chloroplast atpB / E mRNA. A mutation in atp1 results in a failure to accumulate the chloroplast ATP synthase complex due to reduced synthesis of the AtpB subunit. This decrease in AtpB synthesis does not result from a change in atpB mRNA structure or abundance. Instead, the atpB mRNA is associated with abnormally few ribosomes in atp1-1 mutants, indicating that atp1 function is required during translation initiation or early in elongation. Previously, only one nuclear gene that is required for the translation of specific chloroplast mRNAs had been identified in a land plant. Thus, atp1 will be a useful tool for dissecting mechanisms of translational control in chloroplasts. INTRODUCTIONThe chloroplast translation machinery resembles that in eubacteria in many respects (reviewed in Sugiura et al., 1998). Chloroplast ribosomes are similar to bacterial ribosomes in size and antibiotic sensitivities, and the sequences of many chloroplast ribosomal proteins are closely related to those of their prokaryotic ancestors. Despite these similarities, there is increasing evidence that translation mechanisms in chloroplasts differ in several important ways from those in Escherichia coli. For example, chloroplast ribosomes include several proteins that do not have bacterial counterparts (Subramanian, 1993), Shine-Dalgarno sequences play a less prevalent role in chloroplast translation (Sakamoto et al., 1994; Hirose and Sugiura, 1996; Fargo et al., 1998), and the sequence of chloroplast start codons affects translational efficiency but plays only a minor role in positioning the start site (Chen et al., 1995). In addition, there is strong genetic evidence that chloroplast translation is commonly regulated by positively acting regulatory proteins (GoldschmidtClermont, 1998), which is a phenomenon that is rare in E. coli (McCarthy and Gualerzi, 1990).The translation of chloroplast mRNAs is regulated by a variety of factors. For example, light stimulates the translation of both the rbcL and psbA mRNAs (Malnoe et al., 1988;Berry et al., 1990;Staub and Maliga, 1993; Kim et al., 1994) and modulates the rates of both the initiation and elongation steps (Berry et al., 1988(Berry et al., , 1990 Kim et al., 1991; Edhofer et al., 1998). For psbA , this regulation is mediated in part by the 5 Ј untranslated region (UTR) of the mRNA (Staub and Maliga, 1993). Failure to synthesize one component of a photosynthetic complex can influence the translation of other subunits of the same complex: the chloroplast-encoded large subunit of ribulose bisphosphate carboxylase (Rubisco) is translated at reduced rates when synthesis of the nucleusencoded small subunit is disrupted (Khrebtukova and Spreitzer, 1996;Rodermel et al., 1996), and the rate of translation of the petA mRNA is ...

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confidence: 99%

A Nuclear Gene in Maize Required for the Translation of the Chloroplast atpB/E mRNA

McCormac¹,

Barkan²

1999

The Plant Cell

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“…Of the three precursor proteins, only the two with internal His tags were recognized without or with thrombin cleavage (compare lanes 1 and 2 with 5/9 and 6/10, respectively). We found that the signal from the internally tagged proteins was completely lost after incubation with chloroplast lysate, with or without thrombin cleavage (lanes 7,8,11,12). This suggested that the 47 kDa species was created by excising sequences within or upstream of the Sph His tag, and within or downstream of the Bsp His tag, and that the higher molecular weight bands in the splicing reactions all were species lacking the NCL, most likely aggregates with different degrees of resistance to SDS denaturation.…”

Section: Resultsmentioning

confidence: 92%

“…Vivo-All previous reports documenting RB47 purified from chloroplasts described the 47-kDa form, but no evidence for the longer, unspliced form predicted by cDNA (8,11). Many of the analyzed fractions had been highly purified, or were assayed by UV-crosslinking rather than immunoblotting, which might have led to failure to detect the unspliced form.…”

Section: Rb47 Is Present In Unspliced Form Inmentioning

confidence: 99%

Activation of an Endoribonuclease by Non-intein Protein Splicing

Campbell

Stern

2016

Journal of Biological Chemistry

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The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a nonconserved linker (NCL) sequence flanked by highly conserved N-and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions.The chloroplast transcriptome is highly dependent on RNA processing, because pervasive transcription has to be balanced with activities that discard nonfunctional or deleterious transcripts (1). Among the most prominent of these activities are endo-and exoribonucleases that exert RNA quality control and create mature 5Ј and 3Ј termini (2-4). We previously examined the mechanism of 3Ј end maturation for the Chlamydomonas reinhardtii chloroplast atpB mRNA, whose 3Ј terminus is defined by a stem-loop structure. The atpB 3Ј-extended precursor undergoes two-step maturation, in which an endonuclease cleaves at a specific site called the endonuclease cleavage site (ECS) 2 ϳ10 nucleotides (nt) distal to the stem-loop, followed by exonucleolytic trimming (5). Of particular interest was that the sequence downstream of the ECS was extremely rapidly degraded both in vivo and in vitro, even in ectopic contexts (6). We sought to purify this novel ribonuclease activity from Chlamydomonas chloroplasts, with the purification assay based on endoribonucleolytic cleavage near the ECS.During the course of purification, we identified an endoribonuclease activity that appeared to cleave the RNA substrate near the ECS. As shown below, the endoribonuclease we isolated turned out to be RB47, a member of the polyadenylatebinding protein family (PABP). RB47 had been discovered previously in Chlamydomonas chloroplasts through affinity purification of proteins binding to the psbA mRNA 5Ј-UTR (7,8). Based on a variety of correlative experiments, RB47 was proposed to regulate psbA translation in response to light (9 -11) and redox poise (12). RB47, for which the isolated cDNA encoded a protein of 68 kDa (80 kDa by SDS-PAGE), coul...

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“…Physiological studies have shown that both RB47 RNA binding activity and psbA translation are activated in a light-dependent manner (4). Characterization of nuclear mutants lacking RB47 has shown that this protein is required for binding of the complex to the 5Ј-untranslated region (UTR) of the psbA mRNA (6). Absence of RB47 results in the failure of the psbA mRNA to associate with polyribosomes, and hence the loss of translation, suggesting that RB47 acts as a message-specific translation initiation factor for the psbA mRNA (6).…”

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confidence: 99%

“…Characterization of nuclear mutants lacking RB47 has shown that this protein is required for binding of the complex to the 5Ј-untranslated region (UTR) of the psbA mRNA (6). Absence of RB47 results in the failure of the psbA mRNA to associate with polyribosomes, and hence the loss of translation, suggesting that RB47 acts as a message-specific translation initiation factor for the psbA mRNA (6). Binding of the RB47 protein to the 5Ј-UTR of the psbA mRNA can be modulated in vitro by oxidation and reduction reactions (7).…”

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confidence: 99%

Disulfide Bond Formation between RNA Binding Domains Is Used to Regulate mRNA Binding Activity of the Chloroplast Poly(A)-binding Protein

Fong

Lentz

Mayfield

2000

Journal of Biological Chemistry

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Binding of the chloroplast poly(A)-binding protein, RB47, to the psbA mRNA is regulated in response to light and is required for translation of this mRNA in chloroplasts. The RNA binding activity of RB47 can be modulated in vitro by oxidation and reduction. Site-directed mutations to individual cysteine residues in each of the four RNA binding domains of RB47 showed that changing single cysteines to serines in domains 2 or 3 reduced, but did not eliminate, the ability of RB47 to be redoxregulated. Simultaneously changing cysteines to serines in both domains 2 and 3 resulted in the production of RB47 protein that was insensitive to redox regulation but retained the ability to bind the psbA mRNA at high affinity. The poly(A)-binding protein from Saccharomyces cerevisiae lacks cysteine residues in RNA binding domains 2 and 3, and this poly(A)-binding protein lacks the ability to be regulated by oxidation or reduction. These data show that disulfide bond formation between RNA binding domains in a poly(A)-binding protein can be used to regulate the ability of this protein to bind mRNA and suggest that redox regulation of RNA binding activity may be used to regulate translation in organisms whose poly(A)-binding proteins contain these critical cysteine residues.Translational regulation is the predominant mechanism for controlling gene expression in the chloroplast of plants and algae (1-3). In Chlamydomonas reinhardtii, translational regulation of the chloroplast psbA gene, which encodes the D1 protein (a major component of photosystem II), appears to require the binding of a complex composed of four nuclearencoded proteins (4). The 47-kDa member of this protein complex (RB47) has been identified as a polyadenylate-binding protein (PABP) 1 (5). Physiological studies have shown that both RB47 RNA binding activity and psbA translation are activated in a light-dependent manner (4). Characterization of nuclear mutants lacking RB47 has shown that this protein is required for binding of the complex to the 5Ј-untranslated region (UTR) of the psbA mRNA (6). Absence of RB47 results in the failure of the psbA mRNA to associate with polyribosomes, and hence the loss of translation, suggesting that RB47 acts as a message-specific translation initiation factor for the psbA mRNA (6). Binding of the RB47 protein to the 5Ј-UTR of the psbA mRNA can be modulated in vitro by oxidation and reduction reactions (7). The translational machinery of the chloroplast has similarities to prokaryotes including 70 S ribosomes and mRNAs that lack poly(A) tails and 7-methyl-G caps. PABPs have not as yet been identified in prokaryotic organisms. However, the 5Ј-UTR of the psbA mRNA is A/U-rich and contains two stretches of A residues that have been identified as the primary binding site for the RB47 proteins (8). RB47 contains the four RNA recognition motifs (RRMs), which are highly conserved in all PABPs. The structure of RRM1 and RRM2 of the human PABP has been determined (9) and shows a ␤␣␤␤␣␤ secondary structure for each of the RNA binding domain...

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Translation of the Chloroplast psbA mRNA Requires the Nuclear-encoded Poly(A)-binding Protein, RB47

Cited by 73 publications

References 40 publications

A Nuclear Gene in Maize Required for the Translation of the Chloroplast atpB/E mRNA

A Nuclear Gene in Maize Required for the Translation of the Chloroplast atpB/E mRNA

Activation of an Endoribonuclease by Non-intein Protein Splicing

Disulfide Bond Formation between RNA Binding Domains Is Used to Regulate mRNA Binding Activity of the Chloroplast Poly(A)-binding Protein

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