Chloroplast ribonucleoproteins are associated with both mRNAs and intron-containing precursor tRNAs

Tobacco chloroplasts possess five conserved ribonucleoproteins (cpRNPs). To elucidate the function of cpRNPs we analyzed their localization and target nucleic acid molecules in chloroplasts. Immunoprecipitation of the stromal extract and Northern analysis revealed that cpRNPs are associated in vivo with not only various species of chloroplast mRNAs but also intron-containing precursor (pre-) tRNAs. This observation strongly suggests that cpRNPs are involved in RNA processing, including mRNA stability and pre-tRNA splicing.     

Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins.

Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.     

Gene-specific trans-regulatory functions of magnesium for chloroplast mRNA stability in higher plants

In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulated via gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions. psbA mRNA (D1 protein of photosystem II), rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNA(His) gain stability at specific magnesium concentrations in an in vitro degradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulated psbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg(2+) rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability.     

Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine- Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message- specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.     

Chloroplast mRNA 3'' end processing requires a nuclear-encoded RNA-binding protein.

The protein coding regions of plastid mRNAs in higher plants are generally flanked by 3' inverted repeat sequences. In spinach chloroplast mRNAs, these inverted repeat sequences can fold into stem-loop structures and serve as signals for the correct processing of the mature mRNA 3' ends. The inverted repeat sequences are also required to stabilize 5' upstream mRNA segments, and interact with chloroplast protein in vitro. To dissect the molecular components involved in chloroplast mRNA 3' end processing and stability, a spinach chloroplast protein extract containing mRNA 3' end processing activity was fractionated by FPLC and RNA affinity chromatography. The purified fraction consisted of several proteins and was capable of processing the 3' ends of the psbA, rbcL, petD and rps14 mRNAs. This protein fraction was enriched for a 28 kd RNA-binding protein (28RNP) which interacts with both the precursor and mature 3' ends of the four mRNAs. Using specific antibodies to this protein, the poly(A) RNA-derived cDNA for the 28RNP was cloned and sequenced. The predicted amino acid sequence for the 28RNP reveals two conserved RNA-binding domains, including the consensus sequences RNP-CS1 and CS2, and a novel acidic and glycine-rich N-terminal domain. The accumulation of the nuclear-encoded 28RNP mRNA and protein are developmentally regulated in spinach cotyledons, leaves, root and stem, and are enhanced during light-dependent chloroplast development. The general correlation between accumulation of the 28RNP and plastid mRNA during development, together with the result that depletion of the 28RNP from the chloroplast protein extract interferes with the correct 3' end processing of several chloroplast mRNAs, suggests that the 28RNP is required for plastid mRNA 3' end processing and/or stability.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.     

ADP-dependent phosphorylation regulates RNA-binding in vitro: implications in light-modulated translation.

Light-regulated translation of chloroplastic mRNAs in the green alga Chlamydomonas reinhardtii requires nuclear encoded factors that interact with the 5'-untranslated region (5'-UTR) of specific mRNAs to enhance their translation. We have previously identified and characterized a set of proteins that bind specifically to the 5'-UTR of the chloroplastic psbA mRNA. Accumulation of these proteins is similar in dark- and light-grown cells, whereas their binding activity is enhanced during growth in the light. We have identified a serine/threonine protein phosphotransferase, associated with the psbA mRNA-binding complex, that utilizes the beta-phosphate of ADP to phosphorylate and inactivate psbA mRNA-binding in vitro. The inactivation of mRNA-binding in vitro is initiated at high ADP levels, levels that are attained in vivo only in dark-grown chloroplasts. These data suggest that the translation of psbA mRNA is attenuated by phosphorylation of the mRNA-binding protein complex in response to a rise in the stromal concentration of ADP upon transfer of cells to dark.     

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.     

Tubulin mRNA instability and stabilization by protein synthesis inhibitors are reproducible in nontranslating extracts from Chlamydomonas

In Chlamydomonas rein-hardtii, flagellar amputation stimulates an induction in the synthesis of flagellar proteins which allows the cells to rapidly regenerate their flagella. The induction involves the coordinate accumulation and rapid degradation of a large number mRNAs, including those encoding the tubulins. The post-induction degradation of induced tubulin mRNAs has been shown to differ from the consti-tutive turnover pathway in two ways: (1) the rate of degradation is accelerated, and (2) degradation is prevented by inhibition of protein synthesis. In this report, it is shown that the post-induction degradation of all deflagellation-induced mRNAs examined is prevented by cycloheximide (CX), suggesting they all may be degraded via the same pathway. A cell-free decay system has been developed to investigate the degradation pathway. At least two characteristics of tubulir mRNA degradation are reproducible in these extracts: (1) endogenous α-tubulin mRNA is less stable than constitutive mRNAs in the same extract and (2) α-tubulin mRNA in extracts prepared from CX-treated cells (CX ex-tracts) is significantly more stable than it is in extracts from untreated cells (control extracts). This indicates that the mechanism by which CX blocks rapid degradation of tubulin mRNA in vivo is not simply by preventing its translation and suggests the involvement of an altered trans-factor. The difference in tubulin mRNA stability in the two extracts is maintained when the extracts are prepared under conditions that dissociate ribosomes from mRNPs, indicating intact polysome structure is not necessary. Tubulin mRNA-containing polysomes isolated from control and CX extracts are equally stable when assayed alone. However, the poly-somes from control extracts are more sensitive to exogenous RNAse treatment than are those from CX extracts, indicating a structural difference. There are no detectable differences in soluble factors that influence tubulin mRNA degradation rate between control and CX extracts; addition of excess soluble factors to either control or CX extracts does not alter the tubulin mRNA degradation in the extract, nor does a simple one-to-one combination of the two extracts result in stabilization or destabilization of the whole population of tubulin mRNAs in the mixture. The deflagellation-induced mRNAs, as a group, are shown to be particularly susceptible to a nuclease activity in extracts, inhibitable by vanadyl ribonucleoside complexes, which does not appear to attack constitutive mRNAs. It is proposed that a structural difference in the tubulin mRNPs produced in the presence and absence of CX underlies their differences in stabilities, and that a common nuclease targets the induced flagellar protein mRNAs. © 1993 Wiley-Liss, Inc.