An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.     

RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.

An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.     

Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA.

An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain. Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs. It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs. In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases. It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with [32P] orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II. The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein. Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan. RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein. Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast.     

Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding

Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145-1157), we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability.     

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast

A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.     

Control of mRNA stability in chloroplasts by 3'' inverted repeats: effects of stem and loop mutations on degradation of psbA mRNA in vitro.

To investigate the role of mRNA 3' inverted repeats (IRs) in stabilizing plant chloroplast mRNAs, we have measured the processing and stability of wild-type and mutant RNAs corresponding to the 3' end of the spinach chloroplast psbA mRNA. wild-type and mutant 3' IR-RNA precursors were processed at similar rates in a homologous in vitro system, but RNAs with either a mutant loop sequence CUUCGG or a specific base substitution in the IR exhibited an enhanced accumulation of mature product. Incubation of mature products in the in vitro system demonstrated that this was due to an increased stability of the product. These mutant RNAs displayed the same order of stabilities when their decay was measured following electroporation into intact chloroplasts. We found that the in vitro system contains an endonuclease activity that cleaves the wild-type 3' IR-RNA within the loop and also in single-stranded regions, suggesting a possible role for the loop sequence in determining RNA longevity in vitro. Interestingly, the altered loop sequence CUUCGG, which enhances RNA stability in bacteria (1), prolonged the half-life of psbA 3' IR-RNA in vitro and also resulted in an altered endonuclease cleavage pattern. Such nucleases could potentially play an important role in plastid mRNA decay in vivo.     

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.     

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3′-untranslated region stability,processing, and degradation

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3′-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3′-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3′-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3′-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3′-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3′-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3′-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3′-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3′-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.     

Junctions of the large single copy region and the inverted repeats in Spinacia oleracea and Nicotiana debneyi chloroplast DNA: sequence of the genes for tRNAHis and the ribosomal proteins S19 and L2.

This work describes the organization, at the nucleotide sequence level, of genes flanking the junctions of the large single copy regions and the inverted repeats of Spinacia oleracea (spinach) and Nicotiana debneyi chloroplast DNAs. In both genomes, trnH1, the gene for tRNA-His(GUG) is located at the extremity of the large single copy region 3' to psbA, the gene for the 35 kd Photosystem 2 protein. Both psbA and trnH1 are transcribed towards the inverted repeat. In spinach, the first 48 codons of rps19, the gene for the chloroplast ribosomal protein S19, lie in the inverted repeat and the last 44 codons lie in the large single copy region at the end opposite to that carrying trnH1. The gene for a protein homologous to the E. coli ribosomal protein L2, rp12, is in the inverted repeat immediately 5' to rps19 and, like rps19, is transcribed towards the large single copy region. In N. debneyi, but not in spinach, rp12 is interrupted by a 666 bp insertion. The gene for tRNA-lle(CAT), trnl1, is located in the inverted repeats of spinach and N. debneyi, 5' to rp12 and is transcribed in the same direction as rp12.     

5' to 3' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.     

Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions

The mature 3′-end of many chloroplast mRNAs is generated by the processing of the 3′-untranslated region (3′-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3′-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3′-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3′-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3′-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3′-UTRs, finding that each 3′-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3′-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3′-UTR processing activity on ortholog spinach 3′-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3′-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3′-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.