The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Murray P Deutscher - One of the best experts on this subject based on the ideXlab platform.
-
SURVEY AND SUMMARY Exoribonuclease superfamilies: structural analysis and phylogenetic distribution
2020Co-Authors: Murray P DeutscherAbstract:Exoribonucleases play an important role in all aspects of RNA metabolism. Biochemical and genetic analyses in recent years have identified many new RNases and it is now clear that a single cell can contain multiple enzymes of this class. Here, we analyze the structure and phylogenetic distribution of the known Exoribonucleases. Based on extensive sequence analysis and on their catalytic properties, all of the Exoribonucleases and their homologs have been grouped into six superfamilies and various subfamilies. We identify common motifs that can be used to characterize newly-discovered Exoribonucleases, and based on these motifs we correct some previously misassigned proteins. This analysis may serve as a useful first step for developing a nomenclature for this group of enzymes.
-
Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo
Journal of Biological Chemistry, 2012Co-Authors: Tanmay Dutta, Arun Malhotra, Murray P DeutscherAbstract:Abstract Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.
-
Substrate recognition and catalysis by the exoribonuclease RNase R.
Journal of Biological Chemistry, 2006Co-Authors: Helen A. Vincent, Murray P DeutscherAbstract:Abstract RNase R is a processive, 3′ to 5′ hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other Exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3′ overhang is present. Using a variety of specifically designed substrates, we show here that a 3′ overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5′ overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3′ to 5′ polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3′ termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.
-
Exoribonucleases and endoribonucleases
EcoSal Plus, 2004Co-Authors: Zhongwei Li, Murray P DeutscherAbstract:This review provides a description of the known Escherichia coli ribonucleases (RNases), focusing on their structures, catalytic properties, genes, physiological roles, and possible regulation. Currently, eight E. coli Exoribonucleases are known. These are RNases II, R, D, T, PH, BN, polynucleotide phosphorylase (PNPase), and oligoribonuclease (ORNase). Based on sequence analysis and catalytic properties, the eight Exoribonucleases have been grouped into four families. These are the RNR family, including RNase II and RNase R; the DEDD family, including RNase D, RNase T, and ORNase; the RBN family, consisting of RNase BN; and the PDX family, including PNPase and RNase PH. Seven well-characterized endoribonucleases are known in E. coli. These are RNases I, III, P, E, G, HI, and HII. Homologues to most of these enzymes are also present in Salmonella. Most of the endoribonucleases cleave RNA in the presence of divalent cations, producing fragments with 3'-hydroxyl and 5'-phosphate termini. RNase H selectively hydrolyzes the RNA strand of RNA?DNA hybrids. Members of the RNase H family are widely distributed among prokaryotic and eukaryotic organisms in three distinct lineages, RNases HI, HII, and HIII. It is likely that E. coli contains additional endoribonucleases that have not yet been characterized. First of all, endonucleolytic activities are needed for certain known processes that cannot be attributed to any of the known enzymes. Second, homologues of known endoribonucleases are present in E. coli. Third, endonucleolytic activities have been observed in cell extracts that have different properties from known enzymes.
-
Exoribonuclease superfamilies: structural analysis and phylogenetic distribution
Nucleic Acids Research, 2001Co-Authors: Murray P DeutscherAbstract:Exoribonucleases play an important role in all aspects of RNA metabolism. Biochemical and genetic analyses in recent years have identified many new RNases and it is now clear that a single cell can contain multiple enzymes of this class. Here, we analyze the structure and phylogenetic distribution of the known Exoribonucleases. Based on extensive sequence analysis and on their catalytic properties, all of the Exoribonucleases and their homologs have been grouped into six superfamilies and various subfamilies. We identify common motifs that can be used to characterize newly-discovered Exoribonucleases, and based on these motifs we correct some previously misassigned proteins. This analysis may serve as a useful first step for developing a nomenclature for this group of enzymes.
Cecilia M Arraiano - One of the best experts on this subject based on the ideXlab platform.
-
Chapter 5 The Role of 3′–5′ Exoribonucleases in RNA Degradation
Progress in Molecular Biology and Translational Science, 2020Co-Authors: Jose M Andrade, Vânia Pobre, Ines J Silva, Susana Domingues, Cecilia M ArraianoAbstract:RNA degradation is a major process controlling RNA levels and plays a central role in cell metabolism. From the labile messenger RNA to the more stable noncoding RNAs (mostly rRNA and tRNA, but also the expanding class of small regulatory RNAs) all molecules are eventually degraded. Elimination of superfluous transcripts includes RNAs whose expression is no longer required, but also the removal of defective RNAs. Consequently, RNA degradation is an inherent step in RNA quality control mechanisms. Furthermore, it contributes to the recycling of the nucleotide pool in the cell. Escherichia coli has eight 3′–5′ Exoribonucleases, which are involved in multiple RNA metabolic pathways. However, only four Exoribonucleases appear to accomplish all RNA degradative activities: polynucleotide phosphorylase (PNPase), ribonuclease II (RNase II), RNase R, and oligoribonuclease. Here, we summarize the available information on the role of bacterial 3′–5′ Exoribonucleases in the degradation of different substrates, highlighting the most recent data that have contributed to the understanding of the diverse modes of operation of these degradative enzymes.
-
defining the impact of Exoribonucleases in the shift between exponential and stationary phases
Scientific Reports, 2019Co-Authors: Vânia Pobre, Susana Barahona, Tatiane Dobrzanski, M B R Steffens, Cecilia M ArraianoAbstract:: The transition between exponential and stationary phase is a natural phenomenon for all bacteria and requires a massive readjustment of the bacterial transcriptome. Exoribonucleases are key enzymes in the transition between the two growth phases. PNPase, RNase R and RNase II are the major degradative Exoribonucleases in Escherichia coli. We analysed the whole transcriptome of exponential and stationary phases from the WT and mutants lacking these Exoribonucleases (Δpnp, Δrnr, Δrnb, and ΔrnbΔrnr). When comparing the cells from exponential phase with the cells from stationary phase more than 1000 transcripts were differentially expressed, but only 491 core transcripts were common to all strains. There were some differences in the number and transcripts affected depending on the strain, suggesting that Exoribonucleases influence the transition between these two growth phases differently. Interestingly, we found that the double mutant RNase II/RNase R is similar to the RNase R single mutant in exponential phase while in stationary phase it seems to be closer to the RNase II single mutant. This is the first global transcriptomic work comparing the roles of Exoribonucleases in the transition between exponential and stationary phase.
-
Major 3′–5′ Exoribonucleases in the Metabolism of Coding and Non-coding RNA
Progress in Molecular Biology and Translational Science, 2018Co-Authors: Ricardo F. Dos Santos, Cecilia M Arraiano, Ana P Quendera, Sofia Boavida, Andre F Seixas, Jose M AndradeAbstract:Abstract 3′–5′ Exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs and in the maturation of precursor RNAs into their functional forms. The major bacterial 3′–5′ Exoribonucleases responsible for both these activities are PNPase, RNase II and RNase R. These enzymes are of ancient nature with widespread distribution. In eukaryotes, PNPase and RNase II/RNase R enzymes can be found in the cytosol and in mitochondria and chloroplasts; RNase II/RNase R-like enzymes are also found in the nucleus. Humans express one PNPase (PNPT1) and three RNase II/RNase R family members (Dis3, Dis3L and Dis3L2). These enzymes take part in a multitude of RNA surveillance mechanisms that are critical for translation accuracy. Although active against a wide range of both coding and non-coding RNAs, the different 3′–5′ Exoribonucleases exhibit distinct substrate affinities. The latest studies on these RNA degradative enzymes have contributed to the identification of additional homologue proteins, the uncovering of novel RNA degradation pathways, and to a better comprehension of several disease-related processes and response to stress, amongst many other exciting findings. Here, we provide a comprehensive and up-to-date overview on the function, structure, regulation and substrate preference of the key 3′–5′ Exoribonucleases involved in RNA metabolism.
-
Biochemical characterization of Campylobacter jejuni PNPase, an exoribonuclease important for bacterial pathogenicity.
Biochimie, 2018Co-Authors: Jorge Casinhas, Rute G Matos, Nabila Haddad, Cecilia M ArraianoAbstract:Abstract Bacteria need to promptly respond to environmental changes. Ribonucleases (RNases) are key factors in the adaptation to new environments by enabling a rapid adjustment in RNA levels. The exoribonuclease polynucleotide phosphorylase (PNPase) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization in C. jejuni. However, the mechanism of action of this ribonuclease is not yet known. In this work we have characterized the biochemical activity of C. jejuni PNPase. Our results demonstrate that Cj-PNP is a processive 3′ to 5′ exoribonuclease that degrades single-stranded RNAs. Its activity is regulated according to the temperature and divalent ions. We have also shown that the KH and S1 domains are important for trimerization, RNA binding, and, consequently, for the activity of Cj-PNP. These findings will be helpful to develop new strategies for fighting against C. jejuni and may be extrapolated to other foodborne pathogens.
-
characterizing the role of Exoribonucleases in the control of microbial gene expression differential rna seq
Methods in Enzymology, 2018Co-Authors: Vânia Pobre, Cecilia M ArraianoAbstract:Abstract Differential RNA-Seq is a next-generation technology method to determine the significant transcriptomic differences between two and more samples. With this method it is possible to analyze the total RNA content of different samples making it the best global analysis method currently available to study the roles of Exoribonucleases in the cell. These enzymes are responsible for the RNA processing and degradation in the cells and therefore affect the total RNA pool in ways not yet fully understood. In Escherichia coli there are three main degradative Exoribonucleases RNase II, RNase R, and PNPase that degrade the RNA from the 3′ to the 5′-end. These enzymes have several roles in the cell and even though they are degradative enzymes RNase II and PNPase can also protect some RNAs from degradation and PNPase can also act as an RNA polymerase under some conditions. The multiplicity of roles of these Exoribonucleases leads to a very high number of transcripts that are affected by their absence in the cell. With the differential RNA-Seq it is possible to obtain a much deeper understanding of how these enzymes work and regulate the bacterial gene expression. In this chapter we have described a differential RNA-Seq data analysis protocol applied to the study of Exoribonucleases. We also included the protocol for experimental validation of the RNA-Seq data using qPCR and motility assays. Although the methods described in this chapter were applied to the study of the Exoribonucleases, they can also be used for other differential RNA-Seq studies.
Jose M Andrade - One of the best experts on this subject based on the ideXlab platform.
-
Chapter 5 The Role of 3′–5′ Exoribonucleases in RNA Degradation
Progress in Molecular Biology and Translational Science, 2020Co-Authors: Jose M Andrade, Vânia Pobre, Ines J Silva, Susana Domingues, Cecilia M ArraianoAbstract:RNA degradation is a major process controlling RNA levels and plays a central role in cell metabolism. From the labile messenger RNA to the more stable noncoding RNAs (mostly rRNA and tRNA, but also the expanding class of small regulatory RNAs) all molecules are eventually degraded. Elimination of superfluous transcripts includes RNAs whose expression is no longer required, but also the removal of defective RNAs. Consequently, RNA degradation is an inherent step in RNA quality control mechanisms. Furthermore, it contributes to the recycling of the nucleotide pool in the cell. Escherichia coli has eight 3′–5′ Exoribonucleases, which are involved in multiple RNA metabolic pathways. However, only four Exoribonucleases appear to accomplish all RNA degradative activities: polynucleotide phosphorylase (PNPase), ribonuclease II (RNase II), RNase R, and oligoribonuclease. Here, we summarize the available information on the role of bacterial 3′–5′ Exoribonucleases in the degradation of different substrates, highlighting the most recent data that have contributed to the understanding of the diverse modes of operation of these degradative enzymes.
-
Major 3′–5′ Exoribonucleases in the Metabolism of Coding and Non-coding RNA
Progress in Molecular Biology and Translational Science, 2018Co-Authors: Ricardo F. Dos Santos, Cecilia M Arraiano, Ana P Quendera, Sofia Boavida, Andre F Seixas, Jose M AndradeAbstract:Abstract 3′–5′ Exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs and in the maturation of precursor RNAs into their functional forms. The major bacterial 3′–5′ Exoribonucleases responsible for both these activities are PNPase, RNase II and RNase R. These enzymes are of ancient nature with widespread distribution. In eukaryotes, PNPase and RNase II/RNase R enzymes can be found in the cytosol and in mitochondria and chloroplasts; RNase II/RNase R-like enzymes are also found in the nucleus. Humans express one PNPase (PNPT1) and three RNase II/RNase R family members (Dis3, Dis3L and Dis3L2). These enzymes take part in a multitude of RNA surveillance mechanisms that are critical for translation accuracy. Although active against a wide range of both coding and non-coding RNAs, the different 3′–5′ Exoribonucleases exhibit distinct substrate affinities. The latest studies on these RNA degradative enzymes have contributed to the identification of additional homologue proteins, the uncovering of novel RNA degradation pathways, and to a better comprehension of several disease-related processes and response to stress, amongst many other exciting findings. Here, we provide a comprehensive and up-to-date overview on the function, structure, regulation and substrate preference of the key 3′–5′ Exoribonucleases involved in RNA metabolism.
-
major 3 5 Exoribonucleases in the metabolism of coding and non coding rna
Progress in Molecular Biology and Translational Science, 2018Co-Authors: Ricardo Dos F Santos, Cecilia M Arraiano, Ana P Quendera, Sofia Boavida, Andre F Seixas, Jose M AndradeAbstract:Abstract 3′–5′ Exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs and in the maturation of precursor RNAs into their functional forms. The major bacterial 3′–5′ Exoribonucleases responsible for both these activities are PNPase, RNase II and RNase R. These enzymes are of ancient nature with widespread distribution. In eukaryotes, PNPase and RNase II/RNase R enzymes can be found in the cytosol and in mitochondria and chloroplasts; RNase II/RNase R-like enzymes are also found in the nucleus. Humans express one PNPase (PNPT1) and three RNase II/RNase R family members (Dis3, Dis3L and Dis3L2). These enzymes take part in a multitude of RNA surveillance mechanisms that are critical for translation accuracy. Although active against a wide range of both coding and non-coding RNAs, the different 3′–5′ Exoribonucleases exhibit distinct substrate affinities. The latest studies on these RNA degradative enzymes have contributed to the identification of additional homologue proteins, the uncovering of novel RNA degradation pathways, and to a better comprehension of several disease-related processes and response to stress, amongst many other exciting findings. Here, we provide a comprehensive and up-to-date overview on the function, structure, regulation and substrate preference of the key 3′–5′ Exoribonucleases involved in RNA metabolism.
-
Exoribonucleases as modulators of virulence in pathogenic bacteria
Frontiers in Cellular and Infection Microbiology, 2012Co-Authors: Rute G Matos, Vânia Pobre, Jose M Andrade, Catia Barria, Cecilia M ArraianoAbstract:Pathogenic bacteria are responsible for severe diseases worldwide. RNA stability is a major player controlling the expression of virulence factors. Ribonucleases (RNases) are the enzymes responsible for the maturation and degradation of RNA molecules (Arraiano et al., 2010; Silva et al., 2011). Exoribonucleases have been implicated in virulence in an increasing number of pathogens such as Salmonella enterica, Helicobacter pylori, Shigella flexneri, and Aeromonas hydrophila (see Andrade et al., 2009; Matos et al., 2011 and references below). However, the mechanisms underlying virulence are still mostly elusive (Arraiano et al., 2010; Lawal et al., 2011). The recently published paper by Haddad et al. (2012) adds to this list Campylobacter jejuni, one of the most important human foodborne pathogens. Campylobacter is recognized as the leading bacterial cause of gastroenteritis and even more severe clinical manifestations can arise. The present work shows that C. jejuni bacteria lacking an 3′–5′ exoribonuclease called polynucleotide phosphorylase (PNPase) is significantly less virulent than the wild-type strain (Haddad et al., 2012). Different steps have been identified in the ability of different pathogenic bacteria to promote infection, namely motility, adherence, invasion, intracellular replication, or spreading to the neighboring cells. Inactivation of the C. jejuni PNPase is shown to affect many of these steps, with pnp mutants showing distinct phenotypes such as limitations in swimming, substantial delay in the colonization of the chicken gut and a decreased ability to adhere and invade cells. Defects in motility are suggested to be responsible for many of the attenuation of the virulent traits of C. jejuni in the mutant pnp strain. Interestingly, the authors suggest that PNPase may be able to affect flagella-dependent motility by modulation of the NANA synthetase (neuB), involved in the post-translational modification of the flagellin subunit. Furthermore, proteomic studies also showed that PNPase affects the synthesis of proteins involved in virulence, such as LuxS and PEB3. This work confirms the importance of Exoribonucleases, namely PNPase, in cell biology, and virulence (Haddad et al., 2012). Bacterial pathogens rapidly adapt to environmental challenges. Adaptation requires a rapid adjustment in RNA levels, requiring not only transcriptional regulation, but also fine-tuning control of RNA stability. Stress-resistance plays an essential role in the capacity of many pathogenic bacteria to establish and maintain long-term intracellular residence in host cells. Many ribonucleases are regulated by stress conditions, being critical enzymes involved in the adaptation of bacteria to new environmental conditions. In particular, PNPase is a cold-shock protein in Escherichia coli being essential for growth at low temperatures (Zangrossi et al., 2000). Haddad et al. (2009) had previously shown that PNPase was also crucial for C. jejuni growth under cold-shock conditions. This was a relevant discovery especially when considering that this pathogen can persist and grow at refrigerated temperatures. PNPase also seems to be involved in C. jejuni resistance to acidic and oxidative stresses, as the pnp strain shows variations in the levels of the stress-response proteins KatA, DnaK, and Hsp90 (Haddad et al., 2012). In S. enterica, PNPase was shown to be important for acute infection and lethality in a murine model as result of increasing expression of the pathogenicity islands (Clements et al., 2002). In Yersinia pseudotuberculosis and Y. pestis PNPase was shown to be essential for the function of the Yersinia type tree secretion system (TTSS), an organelle that injects effector proteins directly into host cells (Rosenzweig et al., 2007). Interestingly, PNPase has been involved in the post-transcriptional regulation of small non-coding RNAs (Andrade and Arraiano, 2008; De Lay and Gottesman, 2011; Andrade et al., 2012). In C. jejuni, not much is known about this class of regulatory RNAs but transcriptomic studies have identified five candidate regions for harboring sRNAs (Chaudhuri et al., 2011). It is an exciting hypothesis that PNPase is able to regulate small RNAs possibly involved in the virulence traits of C. jejuni although this lacks experimental evidence at the time. Together with PNPase, RNase II, and RNase R are the major Exoribonucleases involved in RNA degradation in E. coli (Figure (Figure1).1). Orthologs have been described in all domains of life (Arraiano et al., 2010). RNase R, a hydrolytic exoribonuclease, is also known to be involved in the virulence of several microorganisms. Like PNPase, RNase R is a cold-shock protein essential for the survival at low temperatures of several microorganisms, such as E. coli, Pseudomonas putida, P. syringae, and A. hydrophila. In some microorganisms RNase R was shown to be necessary for the expression of several invasion factors and mutations on its gene resulted in the reduced expression of virulence phenotypes in S. flexneri and in enteroinvasive E. coli (Tobe et al., 1992). Legionella pneumophila is an intracellular parasite of free-living protozoa which inhabits man-made water distribution systems, and is the most frequent cause of human legionellosis, community-acquired, and nosocomial pneumonia in adults. In this microorganism, RNase R is the only hydrolytic exoribonuclease present. Its activity was shown to be essential for growth and viability at low temperatures and induces competence (Charpentier et al., 2008). Similarly to what was shown in E. coli (Cairrao et al., 2003), RNase R is also a cold-shock protein in A. hydrophila. In this highly toxic microorganism, which is resistant to multiple medications, chlorine, and cold temperatures, RNase R was shown to be essential for viability at lower temperatures and its absence leads to a reduction in motility. The infection of mouse cells with A. hydrophila rnr mutant strains showed that their virulence was attenuated in comparison to the wild-type, which confirms the role of RNase R in pathogenesis (Erova et al., 2008). Figure 1 Schematic representation of the domains found in the Exoribonucleases from PNPase and RNase II families and structures of representative members. (A) Top: PNPase (PDX family) primary structure: two RNase PH catalytic domains followed by two RNA binding ... Considering the important functions that these proteins have in the establishment of virulence, ribonucleases (namely RNase II, RNase R, and PNPase) offer a new perspective for developing efficient compounds in clinical treatments: they can be potential targets to design compounds able to kill specific microorganisms or to reduce their virulence ability. The further study of the function of Exoribonucleases in the control of pathogenesis will certainly help in the comprehension of RNA-related processes involved in infection.
-
Structure and Degradation Mechanisms of 3′ to 5′ Exoribonucleases
Nucleic Acids and Molecular Biology, 2011Co-Authors: Rute G Matos, Michal Malecki, Vânia Pobre, Filipa P Reis, Jose M Andrade, Cecilia M ArraianoAbstract:Exoribonucleases are enzymes that cleave RNA molecules by removing terminal nucleotides from the 3′ or 5′ end of the RNA molecules. They are key factors in RNA metabolism and have a relevant role in the processing and degradation of all types of RNAs. The 3′ to 5′ Exoribonucleases are divided into families, according to their sequence and structural characteristics. The PDX family contains phosphate-dependent degradative enzymes, which can also perform the synthesis of RNA tails when phosphate is limiting. The RNB family contains hydrolytic enzymes with a similar domain organization. All proteins from this widespread family present the characteristic RNB domain responsible for the 3′ to 5′ exoribonuclease activity. In eukaryotes they can act alone or in a complex, the exosome, where they are the only active component. Finally, the DEDD family includes both RNA and DNA exonucleases and they present a similar mechanism of action. In this chapter, we will summarize the available information regarding the 3′ to 5′ Exoribonucleases and discuss their importance for the RNA metabolism.
Jeffrey S. Kieft - One of the best experts on this subject based on the ideXlab platform.
-
Pervasiveness of exoribonuclease-resistant RNAs in plant viruses suggests new roles for these conserved RNA structures
bioRxiv, 2018Co-Authors: Anna-lena Steckelberg, Quentin Vicens, Jeffrey S. KieftAbstract:Exoribonuclease-resistant RNAs (xrRNAs) are discrete folded RNA elements that block the processive degradation of RNA by Exoribonucleases. xrRNAs found in the 3' untranslated regions (UTRs) of animal-infecting flaviviruses and in all three members of the plant-infecting Dianthovirus adopt a complex ring-like fold that blocks the exoribonuclease; this ability gives rise to viral non-coding subgenomic RNAs. The degree to which these folded RNA elements exist in other viruses and in diverse contexts has been unclear. Using computational tools and biochemical assays, we discovered that xrRNA elements are widely found in viruses belonging to the Tombusviridae and Luteoviridae families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Unexpectedly, many xrRNAs are located in intergenic regions rather than in the 3'UTR and some are associated with the 5' ends of subgenomic RNAs with protein-coding potential, suggesting that xrRNAs with similar scaffolds are involved in the maturation or maintenance of diverse subgenomic RNAs, not just the ones generated from the 3'UTR.
-
a folded viral noncoding rna blocks host cell Exoribonucleases through a conformationally dynamic rna structure
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5′ → 3′ cellular Exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping Exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5′ end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use “codegradational remodeling,” exploiting the Exoribonucleases’ degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
-
a folded viral noncoding rna blocks host cell Exoribonucleases through programmed remodeling of rna structure
bioRxiv, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5'-->3' cellular Exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping Exoribonucleases. By solving the structure of a dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the dianthovirus xrRNA can use 'co-degradational remodeling,' exploiting the exoribonuclease's degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
-
Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs.
Nature Communications, 2018Co-Authors: Andrea Macfadden, Zoe O’donoghue, Patrícia A. G. C. Silva, Erich G. Chapman, René C. L. Olsthoorn, Mark G. Sterken, Gorben P. Pijlman, Peter J. Bredenbeek, Jeffrey S. KieftAbstract:Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5′–3′ progression of cellular exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome’s 3′-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse Exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that ‘brace’ against an enzyme’s surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies. Subgenomic flavivirus RNAs are generated by a host exoribonuclease and play an important role in virus replication and pathogenesis. Here, the authors show the mechanism by which subgenomic flavivirus RNAs are generated and identify two structurally distinct sfRNA classes in flaviviruses.
Zhongwei Li - One of the best experts on this subject based on the ideXlab platform.
-
Exoribonucleases and endoribonucleases
EcoSal Plus, 2004Co-Authors: Zhongwei Li, Murray P DeutscherAbstract:This review provides a description of the known Escherichia coli ribonucleases (RNases), focusing on their structures, catalytic properties, genes, physiological roles, and possible regulation. Currently, eight E. coli Exoribonucleases are known. These are RNases II, R, D, T, PH, BN, polynucleotide phosphorylase (PNPase), and oligoribonuclease (ORNase). Based on sequence analysis and catalytic properties, the eight Exoribonucleases have been grouped into four families. These are the RNR family, including RNase II and RNase R; the DEDD family, including RNase D, RNase T, and ORNase; the RBN family, consisting of RNase BN; and the PDX family, including PNPase and RNase PH. Seven well-characterized endoribonucleases are known in E. coli. These are RNases I, III, P, E, G, HI, and HII. Homologues to most of these enzymes are also present in Salmonella. Most of the endoribonucleases cleave RNA in the presence of divalent cations, producing fragments with 3'-hydroxyl and 5'-phosphate termini. RNase H selectively hydrolyzes the RNA strand of RNA?DNA hybrids. Members of the RNase H family are widely distributed among prokaryotic and eukaryotic organisms in three distinct lineages, RNases HI, HII, and HIII. It is likely that E. coli contains additional endoribonucleases that have not yet been characterized. First of all, endonucleolytic activities are needed for certain known processes that cannot be attributed to any of the known enzymes. Second, homologues of known endoribonucleases are present in E. coli. Third, endonucleolytic activities have been observed in cell extracts that have different properties from known enzymes.
-
Exoribonucleases and their multiple roles in rna metabolism
Progress in Nucleic Acid Research and Molecular Biology, 2000Co-Authors: Murray P Deutscher, Zhongwei LiAbstract:Abstract In recent years there has been a dramatic shift in our thinking about ribonucleases (RNases). Although they were once considered to be nonspecific, degradative enzymes, it is now clear that RNases play a central role in every aspect of cellular RNA metabolism, including decay of mRNA, conversion of RNA precursors to their mature forms, and end-turnover of certain RNAs. Recognition of the importance of this class of enzymes has led to an explosion of work and the establishment of significant new concepts. Thus, we now realize that RNases, both endoribonucleases and Exoribonucleases, can be highly specific for particular sequences or structures. It has also become apparent that a single cell can contain a large number of distinct RNases, approaching as many as 20 members, often with overlapping specificities. Some RNases also have been found to be components of supramolecular complexes and to function in concert with other enzymes to carry out their role in RNA metabolism. This review focuses on the Exoribonucleases, both prokaryotic and eukaryotic, and details their structure, catalytic properties, and physiological function.
-
the role of individual Exoribonucleases in processing at the 3 end of escherichia coli trna precursors
Journal of Biological Chemistry, 1994Co-Authors: Zhongwei Li, M P DeutscherAbstract:Abstract We have used an in vitro Escherichia coli tRNA processing system to investigate the specific role of individual Exoribonucleases in the 3' maturation of tRNA precursors. The processing of pre-tRNA(Tyr)su3+ and pre-tRNA(2Arg) was studied using extracts from cells lacking one or multiple Exoribonucleases or using purified RNases. Earlier genetic studies had suggested that multiple Exoribonucleases contributed to the maturation of tRNA precursors, and this was proven directly in the studies described here. Complete 3' processing required the combined action of multiple Exoribonucleases, and each RNase showed distinct specificities for maturation of the different parts of the 3' precursor segment. RNase II and polynucleotide phosphorylase were most effective in shortening long 3' trailer sequences to intermediates with 2-4 extra 3' residues. Final trimming of the last few 3' nucleotides of these precursors was carried out most efficiently by RNases T and PH, but the two enzymes differed in their specificity for individual nucleotide positions. Depending on the tRNA precursor, the relative importance of the various RNases to the overall maturation process differed. We also showed that purified Exoribonucleases can completely complement mutant extracts and that tRNA maturation can be totally reconstructed in vitro using purified enzymes. These studies provide the first detailed information about the specific role of individual Exoribonucleases in tRNA processing, and bring us closer to defining a complete E. coli tRNA maturation pathway.