Polyadenylation and Degradation of mRNA in the Chloroplast1

Chloroplast development is characterized by the synthesis and assembly of the photosynthetic complexes of the thylakoid membranes. This maturation process requires the coordinated expression of many nuclear- and chloroplast-encoded genes. As chloroplasts are semiautonomous organelles, they possess their own genome with its inherent transcriptional and translational machinery. However, nuclear-encoded gene products are also necessary for all of the processes occurring in the chloroplast. In this Update we will focus on the control of chloroplast gene expression at the posttranscription stage. Posttranscriptional processes are widely used in controlling the steady-state levels of plastid mRNAs, and are mediated mainly by nuclear-encoded proteins, suggesting a way in which the nucleus can modulate gene expression in the chloroplast. For example, mutants of nuclear-encoded genes affecting the accumulation of specific chloroplast transcripts have been described in maize, Arabidopsis, and the green algae Chlamydomonas reinhardtii(Goldschmidt-Clermont, 1998). A, RNA metabolism in the chloroplast. Schematic representation of a monocistronic transcription unit in the chloroplast. UTRs are marked with a thin black line; sequences that code for amino acids with a thick black line; and introns with a thick white line. The inverted repeats characterizing the 3′-UTR are symbolized by two arrowheads. A tRNA gene usually presenting 3′ to the inverted repeats is shown as a dashed line. B, The precursor RNA transcribed from this transcription unit undergoes 5′- and 3′-end processing to generate the 5′ and 3′ ends of the mRNA, respectively. C, The 3′ end is located several nucleotides 3′ of the stem-loop structure formed by the inverted-repeats sequence. The introns are removed by splicing, and the tRNA gene is processed by RNaseP. The mRNA is then translated and later degraded. Different aspects of chloroplast mRNA processing and stability have been the subject of recently published reviews (Barkan and Stern, 1998;Drager and Stern, 1998; Goldschmidt-Clermont, 1998; Nickelsen, 1998) and will not be discussed here. We will focus on recent discoveries concerning the molecular mechanism of mRNA polyadenylation and degradation in the chloroplast, and the proteins involved. During leaf development and plastid differentiation, the levels of many plastid mRNAs vary dramatically. The concentration of a specific mRNA is determined by its transcription rate in comparison with its degradation rate. Run-on experiments revealing relative rates of RNA synthesis showed that the different mRNA steady-state levels cannot be related to gene-specific transcriptional activity (Gruissem, 1989). Therefore, changes in the degradation rate of specific mRNAs during chloroplast development occur. To study the degradation of mRNAs in the chloroplast of higher plants in vivo, spinach and barley plants were treated with transcription inhibitors. The rate of decay of chloroplast-encoded mRNAs was determined by quantitative northern analysis using gene-specific probes. The half-life of several chloroplast mRNAs, such as psbA, changed during development from young to mature leaves, whereas that of others, such asrbcL, did not. These results showed that differential mRNA stability contributes to chloroplast mRNA concentrations during leaf development (Klaff and Gruissem, 1991; Kim et al., 1993). Analysis of the transcription, translation, and mRNA levels of 15 plastid genes during barley chloroplast development revealed a dynamic modulation of gene expression and mRNA stability (Rapp et al., 1992). Furthermore, enhanced levels of psbA mRNA in mature barley chloroplasts were due primarily to its selective stabilization. Although data about the RNA elements and the proteins involved in this process are slowly emerging (see below), the precise mechanism by which the stability of a specific chloroplast mRNA changes during plant development and in response to physiological changes (such as light intensity or quality) is still not understood. A central issue for understanding the regulation of mRNA stability is the identification of cis-regulatory elements. These elements are defined by their ability to transfer properties of a certain mRNA to a reporter gene. In general, this is achieved by constructing chimeric genes consisting of the respective RNA element and a reporter gene, such as the Escherichia coli GUS gene, and transforming these constructs into a recipient organism. Chloroplasts of the green algae C. reinhardtii have been routinely transformed, and, recently, techniques for higher plant chloroplast transformation have become available. Nevertheless, most information on cis-regulatory elements in chloroplasts is still derived from C. reinhardtii. In addition, a wide variety of nuclear mutants affecting mRNA stability have been described for C. reinhardtii and higher plants, providing an additional genetic tool for understanding mRNA-degradation pathways (for review, see Goldschmidt-Clermont, 1998). A well-characterized example of an impaired mRNA metabolism in C. reinhardtii is the nuclear mutation nac2-26, which results in the decreased stability of psbD mRNA. Fusing the 5′-UTR of the psbD mRNA to aadA(aminoglycoside adenyltransferase) as a reporter gene showed a destabilized chimeric transcript in the mutant background and normal accumulation in the wild type, reflecting the properties of thepsbD mRNA. These data indicate that the 5′-UTR of the mRNA includes a determinant for psbD mRNA degradation. Instability of the psbD mRNA in the mutant correlates with a 47-kD protein binding to the psbD leader that is present in wild-type C. reinhardtii chloroplasts but not innac2-26 cells, making this protein a candidate for a gene-specific, nuclear-encoded, trans-acting factor that stabilizes the mRNA (Nickelsen et al., 1994). A more complex mechanism for the regulation of mRNA stability has been proposed for the C. reinhardtii petD mRNA. Extensive mutational analysis and experiments using reporter constructs revealed that sequences within the 5′-UTR are essential for translation and affect RNA stability. In all mutants in which translation was compromised, petD mRNA accumulated to a lower level than in wild-type strains, indicating that mRNA stability is not only regulated by RNA-binding proteins but may also be linked to translatability (Sakamoto et al., 1994). The role of nuclear-encoded factors forpetD mRNA stability was confirmed by F16, a nuclear mutant harboring the mutation mcd1-1, which failed to accumulatepetD mRNA. The analysis of this mutant suggested that the mcd1-1 gene product is involved in protein binding the 5′-UTR to prevent digestion of the mRNA by a 5′ to 3′ exonuclease (Drager et al., 1998). In chloroplasts of higher plants information oncis-regulatory elements for mRNA stability has been obtained from mutational studies and from the recent construction of transplastomic tobacco plants. Similar to the situation in C. reinhardtii, no general scheme emerged, but several parameters contributing to the stability of a certain mRNA were observed. Nuclear mutants in which many chloroplast mRNAs were associated with abnormally few ribosomes showed that the level of rbcL mRNA was reduced 4-fold, indicating that the rbcL mRNA is destabilized as a consequence of its decreased polysome association (Barkan, 1993). For the same mRNA, the analysis of constructs consisting of the 5′-UTR fused to a reporter gene showed that mRNA accumulation in the dark is mediated by this region, whereas a leaderless molecule cannot be detected. That study also provided evidence that rbcLmRNA stability is regulated via the 5′-UTR (Shiina et al., 1998). In addition to the 5′-UTR and its role in mRNA accumulation in chloroplasts, the 3′-UTR is also remarkably important. As described above, most chloroplast mRNAs are flanked by a stem-loop structure 3′ of their coding region that takes part in the mature 3′-end processing (for review, see Barkan and Stern, 1998). In addition, these elements are important for impeding the progress of processive exoribonucleases, which can be shown in vitro using a soluble extract from higher plant chloroplasts, and of synthetic RNA fragments as the substrates (Barkan and Stern, 1998; Drager and Stern, 1998). Partial or complete in vivo deletion of the atpB gene stem-loop in transformed C. reinhardtii chloroplasts led to a dramatic decrease in the accumulation of 3′-end-processed mRNA, whereas the transcription rate of this gene remained unaffected. This result indicated that the stem-loop structure is required for the correct 3′-end processing and mRNA accumulation (for review, see Barkan and Stern, 1998; Drager and Stern, 1998). The stem-loop structure can be replaced in vivo by a stretch of 18 guanosines, which also serves as a barrier for a 3′ to 5′ exonuclease in vitro. The correct 3′-end processing of the mRNA mediated by the structural element is nevertheless essential, since it is required for, or strongly stimulates, its translation in C. reinhardtiichloroplasts (Rott et al., 1998). The results summarized in this section suggest that ciselements responsible for the modulation of mRNA stability of specific genes are mainly localized in the 5′-UTR. On the other hand, RNA structural elements located in the 3′-UTR are required for correct processing and are therefore necessary for mRNA stability. We describe below the search for the molecular mechanism of mRNA degradation and the proteins involved. The in vitro RNA processing and degradation system, in which a synthetic RNA is processed or degraded when incubated with soluble chloroplast extract, was utilized to isolate the enzymes involved. The proteins were fractionated using conventional biochemical separation methods, and the purified fractions were analyzed for activity until one or more polypeptides were present. The search for the RNase involved in the 3′-end processing of chloroplast mRNAs yielded 100RNP (100-kD RNA-binding protein; Hayes et al., 1996). Purified 100RNP has biochemical properties similar to PNPase, one of the two exonucleases discovered to date in bacterial cells. Furthermore, the deduced amino acid sequence of the chloroplast 100RNP cDNA was highly homologous to the bacterial PNPase. Does this result imply that the chloroplast RNA processing and degradation system is similar to recently discovered mechanisms in E. coli? (Nierlich and Murakawa, 1996;Carpousis et al., 1999). Together with the discoveries about the mechanisms of mRNA polyadenylation and degradation, which we will describe later, the answer to this question appears to be yes. Nevertheless, unlike bacteria, plastid mRNA metabolism and its associated enzymes are controlled by the nucleus and may be regulated by light or by the redox state of the chloroplast (Hayes et al., 1996). The E. coli RNA degradosome is a multienzyme complex consisting of the exoribonuclease PNPase, the endonuclease RNase E, a DEAD-box ATP-dependent RNA-helicase, and the enzyme enolase (Carpousis et al., 1999). This high-molecular-mass protein complex is important in RNA processing and mRNA degradation in the bacterial cell, since two of its components, PNPase and RNase E, have been shown to be key elements in these processes. The chloroplast 100RNP/PNPase was isolated in a high-molecular-mass complex of about 600 kD. A 67-kD protein cross-reacting with antibodies prepared against RNase E of E. coli and displaying endoribonuclease activity was copurified with that complex (Hayes et al., 1996). Therefore, it is tempting to suggest that a complex similar to the bacterial degradosome exists in the chloroplast, preserving its ancestral prokaryotic origin (Carpousis et al., 1999). In both bacteria and chloroplasts, it appears that not all of the PNPase population is associated with the degradosome (Carpousis et al., 1994; Lisitsky et al., 1997b). The question of whether different forms of the 100RNP/PNPase are involved in each RNA metabolic activity, such as 3′-end processing and degradation, therefore, remains open. In vitro experiments using synthetic RNAs and purified 100RNP/PNPase have shown much higher enzyme activity on polyadenylated RNA (see below). This selectivity to polyadenylated RNA resulted from the high-affinity binding of the 100RNP/PNPase to poly(A) sequences (Lisitsky et al., 1997b). It is interesting that a similar function and mode of action have recently been reported for the E. coli PNPase and for another exoribonuclease, RNase II (Coburn and Mackie, 1996; Lisitsky and Schuster, 1999). Therefore, identification, isolation, and characterization of the other chloroplast exoribonucleases will determine whether the preference for poly(A)-rich RNAs is only intrinsic to the 100RNP/PNPase, or if it is shared by several or all of the chloroplast exonucleases. In addition, a protein complex composed of several RNA-degradation enzymes was recently identified and isolated in yeast, and was named the exosome (Mitchel et al., 1997;Jacobs et al., 1998). Are the bacterial degradosome, the chloroplast degradosome, and the yeast cytoplasm exosome related to each other functionally and/or evolutionarily? This interesting question is now under intensive study. Posttranscriptional addition of a poly(A) tail to the 3′ end of mRNA was first identified and characterized in eukaryotic cells for viral and nuclear-encoded mRNAs. In these cells the poly(A) tails are formed by the addition of about 250 adenylate residues to a 3′ end generated by endonucleolytic cleavage of the precursor RNA. A complex assembly of proteins is required, along with the activity of poly(A) polymerase. The result of this process is that most of the mRNA are polyadenylated and 1996). is the function of the poly(A) studies have revealed that the poly(A) tail of eukaryotic nuclear-encoded mRNAs is an important determinant of their maturation and of to we the transfer of the precursor RNA from the nucleus to the cytoplasm and the of stability. In yeast, is a in the degradation of nuclear-encoded mRNAs. The poly(A) tail and the protein that to it were to be important for the of the translation can the poly(A) located in the 3′ end of the RNA molecule control the translation at the 5′ A mRNA has been proposed the mRNA 5′ and 3′ ends can with each In this the protein that is associated with the poly(A) tail the binding of the to mRNA by with the translation factor which also to and the 5′ of mRNA. The of the mRNA in this way is required for the of translation et al., For a polyadenylation was to be associated with eukaryotic mRNAs. such as and RNAs in were not to be of these RNA not have a poly(A) tail in their 3′ Nevertheless, poly(A) tails have recently been in bacteria The polyadenylated RNA for only a of the population of the same RNA in the This in mutant bacteria cells that On the other hand, in mutants in which RNA polyadenylation was due to the of poly(A) the half-life of the RNA these results imply unlike the nucleus and cytoplasm of eukaryotic cells, the poly(A) tail is important for the and translation of mRNA and the of the poly(A) tail in part of the mRNA degradation the addition of poly(A) tails in bacterial mRNAs their degradation. polyadenylation of RNA in bacteria cells is a part of the molecular mechanism of RNA degradation in polyadenylation required for RNA degradation, or is an additional degradation in the the degradation specific to certain of RNA such as is the sequence of in this RNA-degradation and is the These are now under intensive et al., 1999). Chloroplasts from that were into eukaryotic cells in an of the gene expression of the chloroplast of the However, of the gene expression in the chloroplast are similar to the eukaryotic For example, chloroplast genes are usually with introns that have not been in The question then RNA polyadenylation in the it similar to the eukaryotic nuclear-encoded such as bacteria, or is it a It is interesting to that poly(A) RNA was in the chloroplast more than and experiments with RNA from it was determined that about of the RNA to and that the chloroplast poly(A) about nucleotides in Nevertheless, the situation in the polyadenylation of bacterial polyadenylation has been as a of eukaryotic nuclear and viral mRNAs. However, polyadenylation of prokaryotic and mRNAs has recently as the focus of as part of the mRNA degradation To polyadenylated RNA in the chloroplast, the of was RNA was isolated from purified chloroplasts and cDNA was from the polyadenylated RNA The cDNA to a specific gene was using a gene-specific on one and a tail on the other et al., et al., 1996). the sequences of the poly(A) tails of chloroplast RNAs revealed several interesting with poly(A) tails in bacteria and yeast, the chloroplast poly(A) tails are tails of nucleotides were with only to nucleotides in bacteria and unlike eukaryotic nuclear-encoded and bacterial the poly(A) in the chloroplast was not to be a of but was composed of of by guanosines, and, on by and A chloroplast poly(A)-rich tail usually guanosines, and and making sequences (Lisitsky et al., 1996). are the poly(A) tails in the chloroplast but not in nuclear-encoded or bacteria a function for this or it of the chloroplast poly(A) we not the to these nucleotides in addition to were recently in RNA isolated from E. coli cells under and The is related to the of the polyadenylation in the RNA molecule and will be discussed for the of polyadenylated are transcription and cleavage of RNA. of the polyadenylation that were by of chloroplast RNA were localized within the amino region of the mRNA. of mRNA polyadenylated at the 3′ end were also but were at lower in (Lisitsky et al., 1996). This result indicated that most mRNAs are did the RNA the addition of poly(A)-rich tails The may from transcription or cleavage of a used to polyadenylation of mRNA. The mRNA and are symbolized by thin and the coding region by a thick black line. of purified chloroplast RNA using the and revealed the poly(A)-rich sequences were to the 3′ of the cleavage product The 5′ of the cleavage product was determined by analysis the of RNA in the chloroplast mRNA degradation system (see was to be one 3′ to Therefore, polyadenylation at the 3′ end of the endonucleolytic cleavage Similar of mRNA degradation by polyadenylation and the chloroplasts were incubated for or in the or of or yeast tRNA as in the RNA was from of chloroplasts and analyzed by an RNA that was with a The of is indicated on the as the of nucleotides in vitro polyadenylation system was used to the of mRNA polyadenylation and degradation to isolate the proteins and to their In RNAs to the chloroplast RNAs be polyadenylated at their 3′ end using a soluble chloroplast protein extract by the addition of (Lisitsky et al., 1996). In vitro analysis of chloroplast polyadenylation activity revealed to and reflecting the of the poly(A)-rich tails by described In this it is interesting to that and were purified from chloroplasts and Furthermore, in vitro polyadenylation activity is on the RNAs were polyadenylated in a highly with the stem-loop structure of the mature plastid mRNA 3′ end (Lisitsky et al., 1996). this is in with the polyadenylated RNA at the mature 3′ end by a stem-loop were than at the of the RNA RNA can be in a using a of the sequence of the gene and an RNA from a RNA degradation by chloroplast proteins can be by a chloroplast soluble protein extract harboring the as as the other RNA-binding proteins and of this with a synthetic RNA. In synthetic polyadenylated RNA was degraded with the same RNA incubated in a soluble chloroplast protein experiments revealed that polyadenylated RNA are more for the degradation than These results suggest that poly(A)-rich tails a role in the degradation of products of mRNA decay in the chloroplast by the cleavage products for degradation, due to their to chloroplast et al., 1996; Lisitsky et al., A for the situation in the chloroplast is that the relative concentration of the exonucleases is such that they are all in polyadenylated endonucleolytic cleavage In this only polyadenylated RNA will be as was described for the experiments using polyadenylation in the chloroplast The 100RNP/PNPase discussed above, similar to the bacterial PNPase, is a processive exoribonuclease binding to the 3′ the RNA by from the As described above, this protein be obtained as a purified Therefore, it was to determine whether the purified enzyme the degradation activity to polyadenylated RNA with the chloroplast protein The other was that other proteins are In experiments using purified 100RNP/PNPase, the polyadenylated RNA with the RNA for the as shown for the soluble protein extract (Lisitsky et al., 1997b). The results that for polyadenylated RNA is an intrinsic of the enzyme as one Therefore, for polyadenylated RNA not on the association with the the degradosome described the activity to polyadenylated RNA due to the higher binding activity of this protein to a poly(A)-rich sequence or to the degradation activity of polyadenylated of the 100RNP/PNPase to as as to other RNA higher binding of this protein to poly(A) than to other RNA (Lisitsky et al., 1997b). On the other hand, the degradation rate was similar for all RNA These results suggest that the degradation of polyadenylated RNA in the chloroplast is on the exoribonuclease binding to the poly(A) sequence. This a poly(A) high-affinity binding in addition to the RNA degradation In addition, the be that chloroplast also polyadenylated RNA with higher than RNA. in vitro degradation activity of bacterial RNase II to polyadenylated RNA was recently (Coburn and Mackie, 1996). the E. coli PNPase was recently to polyadenylated RNA with higher than other RNA (Lisitsky and Schuster, 1999). A for the degradation of mRNA in the chloroplast. A, Schematic representation of the The white the amino region, and the stem-loop inverted repeats in the 5′- and that stem-loop B, The in the mRNA degradation process is suggested to be by an The endonuclease is symbolized by C, A poly(A)-rich which can be to several nucleotides in is then to the 3′ end of the 5′ The polyadenylated RNA molecule is degraded by an the The two mechanisms for the half-life of a RNA The first that the mRNA molecule is it is for the degradation In this the is the endonucleolytic this has mRNA will be polyadenylated and degraded. In such a the and modulation of activity and/or expression of the determine the half-life of a mRNA and the of the poly(A) and exonuclease not control the The that other in the degradation the degradation rate. It is still if the poly(A) tail which can to several the degradation rate. Furthermore, the residues of the poly(A) tails of chloroplast psbA mRNA may be involved in the activity of the respective On the other hand, they may the of the poly(A) and Lisitsky et al., 1996). In vitro experiments in which synthetic transcribed RNA with poly(A) tails of different and different of residues were incubated with chloroplast protein extract revealed in degradation The of these results in to the in vivo situation is still and not the endonucleolytic cleavage product is degraded. is that many endonucleolytic along the mRNA until the RNA the 3′-UTR is degraded by the by the stem-loop A of polyadenylation of the mature 3′ end has been obtained in vivo and in vitro (Lisitsky et al., 1996). an additional degradation in the chloroplast that is of polyadenylation may For example, evidence of 5′ to 3′ exonuclease activity was recently obtained in C. reinhardtii chloroplasts (Drager et al., 1998). or not an additional degradation the results of the experiments using the polyadenylation indicated that the rbcL and psbA mRNAs are degraded only in the degradation To the role of this degradation in the regulation of plastid mRNA the degradation and the for polyadenylation in and has to be In these the psbA RNAs have and are degraded. the few understanding of the chloroplast mRNA degradation has For certain mRNAs, such as psbA, the different of its specific decay have been The of endonucleolytic degradation is of that of the prokaryotic This is by the that one of the enzymes involved has been identified as structural and with its prokaryotic The chloroplast, is in part regulated by the nucleus and by such as the of its mRNA the properties of plants than of Therefore, the chloroplast an by these different the biochemical of plastid mRNA degradation, will on the regulation of mRNA stability as part of the leaf development and to This is to on We to the of the and for and We also to for transcription