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.     

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.     

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.     

Multiple determinants of eukaryotic mRNA stability

Regulated changes in mRNA stability play an important role in modulating the level of expression of many eukaryotic genes. In several systems, specific sequence determinants that dictate mRNA instability have been identified. Thus, the presence of instability determinants, and not the absence of sequences that dictate stability, appears to be required for regulated mRNA degradation. These instability determinants presumably interact with specific nucleases or other trans-acting factors that regulate the accessibility of the domain to nucleases. Although each RNA destabilization pathway has unique features, in many cases RNA degradation requires ongoing protein synthesis. In some of the systems discussed, the mRNAs are degraded co-translationally, perhaps by a ribosome-associated ribonuclease. For other messages, the mechanistic reasons for the dependence of mRNA degradation on ongoing protein synthesis are still unknown.     

Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method

Polyelectrolyte complexes between cationic polymers and DNA have emerged as potential nonviral vectors for DNA delivery. For successful in vivo delivery, methods for analyzing their ability to prevent digestion of the DNA payload by serum nucleases are essential. We report here a simple assay to determine degradation of DNA in these complexes using standard electrophoretic techniques. The assay is based on a high pH buffer which can dissociate the complexes under standard electrophoretic conditions. This assay can be used qualitatively to determine the time taken for degradation to occur. Alternatively, with a standard gel analysis program it can be used quantitatively to investigate rates of DNA degradation from complexes in the presence of serum nucleases. We have shown that it can distinguish between different formulations with the same polymer, and also to distinguish between the time taken to degradation and the rates of degradation of DNA in complexes formed with two structurally related, linear polyamidoamine polymers. The assay could also distinguish between the time to degradation using poly-l-lysine complexes, although these were less well dissociated by the electrophoresis buffer, and could not be analyzed quantitatively. This assay will be of value in investigating and developing polyelectrolyte formulations for parenteral administration.     

Cuts can kill: the roles of apoptotic nucleases in cell death and animal development

Chromosome fragmentation is one of the major biochemical hallmarks of apoptosis. However, until recently, its roles in apoptosis and mechanisms of action remained elusive. Recent biochemical and genetic studies have shown that chromosome fragmentation is a complex biochemical process that involves a plethora of conserved nucleases with distinct nuclease activities and substrate specificities. These apoptotic nucleases act cooperatively among themselves and with other nonnuclease cofactors to promote stepwise chromosome fragmentation and DNA degradation. Importantly, in addition to its direct contribution to the dismantling of the dying cell, apoptotic DNA degradation can facilitate cell killing and other apoptotic events such as clearance of apoptotic cells. Furthermore, some apoptotic nucleases apparently affect other aspects of animal development, including immune responses. The identification of new apoptotic nucleases and analysis of their functions in apoptosis and animal development should pave the way for future studies to uncover new functions for apoptotic nucleases and shed light on the hidden links between apoptotic DNA degradation and human diseases.     

Variations in the enzymatic activity of S1-type nucleases results from differences in their active site structures

BackgroundS1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn²⁺-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities.MethodsS1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay.ResultsWe discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues.ConclusionsOur findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases.General significanceOur findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family.     

Functional genomic analysis of apoptotic DNA degradation in C. elegans

Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Using an RNAi-based functional genomic approach, we have identified seven additional cell death-related nucleases (crn genes), which along with two known nucleases (CPS-6 and NUC-1) comprise at least two independent pathways that contribute to cell killing, and likely signaling for phagocytosis, by degrading chromosomal DNA. Several crn genes have human homologs that are important for RNA processing, protein folding, DNA replication, and DNA damage repair, suggesting dual roles for CRN nucleases in cell survival and cell death. It should now be possible to systematically decipher the mechanisms of apoptotic DNA degradation.