Multiple instability-regulating sites in the 3'' untranslated region of the urokinase-type plasminogen activator mRNA.

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.     

Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions

The functional stability of mRNA is one of the crucial factors affecting the efficiency of cell-free protein synthesis. The importance of the stability of mRNA in the prolonged synthesis of protein molecules becomes even greater when the cell-free protein synthesis is directed by PCR-amplified DNAs, because the linear DNAs are rapidly degraded by the endogenous nucleases and, thus, the continuous generation of mRNA molecules is limited. With the aim of developing a highly efficient cell-free protein synthesis system directed by PCR products, in this study, we describe a systematic approach to enhance the stability of mRNA in cell-free extracts. First, exonuclease-mediated degradation was substantially reduced by introducing a stem-loop structure at the 3'-end of the mRNA. The endonucleolytic cleavage of the mRNA was minimized by using an S30 extract prepared from an Escherichia coli strain that is deficient in a major endonuclease (RNase E). Taken together, through the retardation of the endonucleolytic and exonucleolytic degradations of the mRNA molecules, the level of protein expression from the PCR-amplified DNA templates becomes comparable to that of conventional plasmid-based reactions. The enhanced productivity of the PCR-based cell-free protein synthesis enables the high-throughput generation of protein molecules required for many post-genomic applications.     

Proteome analysis of chloroplast mRNA processing and degradation

Chloroplasts have a complex enzymatic machinery to adjust the relative half-life of their mRNAs to environmental signals. Soluble protein extracts from spinach (Spinacia oleracea L.) chloroplasts that correctly reproduce in vitro the differential mRNA stability observed in vivo were analyzed using shotgun proteomics to identify the proteins that are potentially involved in this process. The combination of a novel strategy for the database-independent detection of proteins from MS/MS data with standard database searches allowed us to identify 243 proteins with high confidence, which include several nucleases and RNA binding proteins but also proteins that have no reported function in chloroplast mRNA metabolism. Characterization of enzyme activities that adjust mRNA stability in response to illumination revealed that the dark-induced RNA degradation pathway involves enzymatic activities that differ from those that direct RNA processing and stabilization in the light. Dark-induced mRNA degradation comprises a MgCl2-independent and a MgCl2-dependent step, which releases nucleoside di- and monophosphates from the petD 3'-UTR precursor substrate. RNA degradation can be blocked with RNasin, a potent inhibitor of eukaryotic ribonucleases, suggesting that chloroplast mRNA degradation involves enzymes that are distinct from those found in prokaryotic-type RNA degradation. On the basis of the identified proteins and the in vitro characterization of the RNA degradation activities, we discuss scenarios and components that potentially determine plastid mRNA stability.     

mRNA degradation in bacteria

Messenger RNAs in prokaryotes exhibit short half-lives when compared with eukaryotic mRNAs. Considerable progress has been made during recent years in our understanding of mRNA degradation in bacteria. Two major aspects determine the life span of a messenger in the bacterial cell. On the side of the substrate, the structural features of mRNA have a profound influence on the stability of the molecule. On the other hand, there is the degradative machinery. Progress in the biochemical characterization of proteins involved in mRNA degradation has made clear that RNA degradation is a highly organized cellular process in which several protein components, and not only nucleases, are involved. In Escherichia coli, these proteins are organized in a high molecular mass complex, the degradosome. The key enzyme for initial events in mRNA degradation and for the assembly of the degradosome is endoribonuclease E. We discuss the identified components of the degradosome and its mode of action. Since research in mRNA degradation suffers from dominance of E. coli-related observations we also look to other organisms to ask whether they could possibly follow the E. coli standard model.     

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.     

Quality control systems for aberrant mRNAs induced by aberrant translation elongation and termination

RNA processing is an essential gene expression step and plays a crucial role to achieve diversity of gene products in eukaryotes. Various aberrant mRNAs transiently produced during RNA processing reactions are recognized and eliminated by specific quality control systems. It has been demonstrated that these mRNA quality control systems stimulate the degradation of aberrant mRNA to prevent the potentially harmful products derived from aberrant mRNAs. Recent studies on quality control systems induced by abnormal translation elongation and termination have revealed that both aberrant mRNAs and proteins are subjected to rapid degradation. In NonStop Decay (NSD) quality control system, a poly(A) tail of nonstop mRNA is translated and the synthesis of poly-lysine sequence results in translation arrest followed by co-translational degradation of aberrant nonstop protein. In No-Go Decay (NGD) quality control system, the specific amino acid sequences of the nascent polypeptide induce ribosome stalling, and the arrest products are ubiquitinated and rapidly degraded by the proteasome. In Nonfunctional rRNA Decay (NRD) quality control system, aberrant ribosomes composed of nonfunctional ribosomal RNAs are also eliminated when aberrant translation elongation complexes are formed on mRNA. I describe recent progresses on the mechanisms of quality control systems and the relationships between quality control systems. This article is part of a Special issue entitled: RNA Decay mechanisms.     

Capping strategies in RNA viruses

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.     

Generation of Temperature-Sensitive Cbp1 Strains of Saccharomyces Cerevisiae by Pcr Mutagenesis and in Vivo Recombination: Characteristics of the Mutant Strains Imply That Cbp1 Is Involved in Stabilization and Processing of Cytochrome B Pre-mRNA

Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.     

Probing the substrate specificity of Escherichia coli RNase E using a novel oligonucleotide-based assay

Endoribonuclease RNase E has a central role in both processing and decay of RNA in Escherichia coli, and apparently in many other organisms, where RNase E homologs were identified or their existence has been predicted from genomic data. Although the biochemical properties of this enzyme have been already studied for many years, the substrate specificity of RNase E is still poorly characterized. Here, I have described a novel oligonucleotide-based assay to identify specific sequence determinants that either facilitate or impede the recognition and cleavage of RNA by the catalytic domain of the enzyme. The knowledge of these determinants is crucial for understanding the nature of RNA–protein interactions that control the specificity and efficiency of RNase E cleavage and opens new perspectives for further studies of this multi-domain protein. Moreover, the simplicity and efficiency of the proposed assay suggest that it can be a valuable tool not only for the characterization of RNase E homologs but also for the analysis of other site-specific nucleases.     

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.     

Increased messenger RNA from protein synthesis inhibited human fibroblasts

Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as “superinduction” and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)-cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal ³H-uridine labeled RNA, a 1.5- to 2-fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.     

Polyadenylation accelerates degradation of chloroplast mRNA.

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.     

La Crosse virus infection of mammalian cells induces mRNA instability.

La Crosse virus infection of BHK cells leads to a dramatic shutoff of not only host protein synthesis but also viral protein synthesis later in infection. This shutoff can be accounted for by the loss of the cytoplasmic cellular and viral mRNAs. The induction of mRNA instability requires extensive virus replication, since when cycloheximide is added early in infection the preexisting viral and cellular mRNAs do not decrease upon incubation of the cultures. Pretreatment of the cultures with actinomycin D does not affect the ability of La Crosse virus infection to induce mRNA instability, and examination of the rRNAs shows no evidence of specific degradation due to activation of the interferon-associated latent RNase. The induction of mRNA instability therefore does not appear to operate through an interferon pathway. Viral mRNA synthesis, on the other hand, is not turned off during infection, and the cap-dependent endonuclease involved in viral mRNA initiation may be responsible for the mRNA instability.     

A single-stranded nucleic acid-binding protein from Artemia salina. I. Purification and characterization

A protein that binds tightly to single-stranded but not to double-strained nucleic acids has been purified to homogeneity from a high salt wash of ribosomes from cryptobiotic Artemia saline gastrulae. The protein, designated HD40 to indicate a helix-destabilizing protein with a molecular weight of 40,000, is present in the high-salt ribosomal wash at a level of about 2 molecules per 80 S ribosome. The protein is monomeric at salt concentrations from 0.01 to 0.5 M and has an alpha-helix content of approximately 15%. The amino acid composition of HD40 is characterized by a high glycine content (19.5 mol%), the absence of cysteine, and the presence of the unusual amino acid dimethylarginine. The isolated protein binds preferentially to natural RNA over denatured DNA. HD40 inhibits protein synthesis directed by poly(rU) and by Artemia poly(A+) RNA in cell-free systems derived from Artemia and from wheat germ; inhibition is relieved by excess of mRNA. Single-stranded ribo- and deoxyribopolynucleotides are largely protected from degradation by nucleases when complexed with HD40.