Gadi Schuster

HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs

The psbB-psbT-psbH-petB-petD operon of higher plant chloroplasts is a heterogeneously composed transcriptional unit that undergoes complex RNA processing events until the mature oligocistronic RNAs are formed. To identify the nucleus-encoded factors required for the processing and expression of psbB-psbT-psbH-petB-petD transcripts, we performed mutational analysis using Arabidopsis. The allelic nuclear mutants hcf152-1 and hcf152-2 were identified that are affected specifically in the accumulation of the plastidial cytochrome b6f complex. In both mutants, reduced amounts of spliced petB RNAs (encoding the cytochrome b6 subunit) were detected, thus explaining the observed protein deficiencies. Additionally, mutant hcf152-1 is affected in the accumulation of transcripts cleaved between the genes psbH and petB. As a result of a close T-DNA insertion, the HCF152 gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF152 encodes a pentatricopeptide repeat (PPR) protein with 12 putative PPR motifs that is located inside the chloroplast. The protein shows a significant structural, but not primary, sequence similarity to the maize protein CRP1, which is involved in the processing and translation of the chloroplast petD and petA RNAs. In addition, we found that HCF152 is an RNA binding protein that binds certain areas of the petB transcript. The protein possibly exists in the chloroplast as a homodimer and is not associated with other proteins to form a high molecular mass complex.

Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein

Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI–atpH intergenic region (i) blocks both 5′→3′ and 3′→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5′-terminus in conjunction with a generic 5′→3′ exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10’s ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins.

Processing and degradation of chloroplast mRNA.

The conversion of genetic information stored in DNA into a protein product proceeds through the obligatory intermediate of messenger RNA. The steady-state level of an mRNA is determined by its relative synthesis and degradation rates, i.e., an interplay between transcriptional regulation and control of RNA stability. When the biological status of an organism requires that a gene products abundance varies as a function of developmental stage, environmental factors or intracellular signals, increased or decreased RNA stability can be the determining factor. RNA stability and processing have long been known as important regulatory points in chloroplast gene expression. Here we summarize current knowledge and prospects relevant to these processes, emphasizing biochemical data. The extensive literature on nuclear mutations affecting chloroplast RNA metabolism is reviewed in another article in this volume (Barkan and Goldschmidt-Clermont, this issue).

Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

ABSTRACT The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli,polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of Piand ADP. As expected of a phosphorylase, Pi enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the completeArabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

Polyadenylation and Degradation of Human Mitochondrial RNA: the Prokaryotic Past Leaves Its Mark

ABSTRACT RNA polyadenylation serves a purpose in bacteria and organelles opposite from the role it plays in nuclear systems. The majority of nucleus-encoded transcripts are characterized by stable poly(A) tails at their mature 3′ ends, which are essential for stabilization and translation initiation. In contrast, in bacteria, chloroplasts, and plant mitochondria, polyadenylation is a transient feature which promotes RNA degradation. Surprisingly, in spite of their prokaryotic origin, human mitochondrial transcripts possess stable 3′-end poly(A) tails, akin to nucleus-encoded mRNAs. Here we asked whether human mitochondria retain truncated and transiently polyadenylated transcripts in addition to stable 3′-end poly(A) tails, which would be consistent with the preservation of the largely ubiquitous polyadenylation-dependent RNA degradation mechanisms of bacteria and organelles. To this end, using both molecular and bioinformatic methods, we sought and revealed numerous examples of such molecules, dispersed throughout the mitochondrial genome. The broad distribution but low abundance of these polyadenylated truncated transcripts strongly suggests that polyadenylation-dependent RNA degradation occurs in human mitochondria. The coexistence of this system with stable 3′-end polyadenylation, despite their seemingly opposite effects, is so far unprecedented in bacteria and other organelles.

Polyadenylation of ribosomal RNA in human cells

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3′ ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT–PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.

Dis3-like 1: a novel exoribonuclease associated with the human exosome.

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine‐subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome‐associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity‐purified human exosome complexes identified a novel exosome‐associated exoribonuclease, human Dis3‐like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co‐immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA‐mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)‐tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome‐associated exoribonuclease in the cytoplasm of human cells.

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22‐kd heat‐shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)+RNA, using the wheat‐germ and reticulocyte lysate systems. Heat‐shock treatment of pea plants for 2 h at 42‐45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22‐kd protein is synthesized as a 26‐kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post‐translational transport into intact chloroplasts in vitro of the 26‐kd precursor, the protein is processed but the resulting 22‐kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat‐shocked plants, the 22‐kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)+RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre‐existing poly(A)+RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)−RNAs persist and are translated. As in pea, a poly(A)+RNA coded 22‐kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22‐kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)+RNA coding for the 22‐kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat‐induced proteins is identical to the light‐inducible proteins of the chloroplast membranes.

RNA polyadenylation in Archaea : not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.

Chapter 10 RNA Polyadenylation and Decay in Mitochondria and Chloroplasts

Mitochondria and chloroplasts were originally acquired by eukaryotic cells through endosymbiotic events and retain their own gene expression machinery. One hallmark of gene regulation in these two organelles is the predominance of posttranscriptional control, which is exerted both at the gene-specific and global levels. This review focuses on their mechanisms of RNA degradation, and therefore mainly on the polyadenylation-stimulated degradation pathway. Overall, mitochondria and chloroplasts have retained the prokaryotic RNA decay system, despite evolution in the number and character of the enzymes involved. However, several significant differences exist, of which the presence of stable poly(A) tails, and the location of PNPase in the intermembrane space in animal mitochondria, are perhaps the most remarkable. The known and predicted proteins taking part in polyadenylation-stimulated degradation pathways are described, both in chloroplasts and four mitochondrial types: plant, yeast, trypanosome, and animal.