Creative catalysis: pieces of the RNA world jigsaw

Novel ribozymes produced by in vitro selection techniques provide insights into the possible mechanisms of protein synthesis evolution. The availability of such ribozymes also paves the way for experiments to explore the evolution of RNA–protein enzymes.     

The roads to and from the RNA world

The historical existence of the RNA world, in which early life used RNA for both genetic information and catalytic ability, is widely accepted. However, there has been little discussion of whether protein synthesis arose before DNA or what preceded the RNA world (i.e. the pre-RNA world). We outline arguments of what route life may have taken out of the RNA world: whether DNA or protein followed. Metabolic arguments favor the possibility that RNA genomes preceded the use of DNA as the informational macromolecule. However, the opposite can also be argued based on the enhanced stability, reactivity, and solubility of 2-deoxyribose as compared to ribose. The possibility that DNA may have come before RNA is discussed, although it is a less parsimonious explanation than DNA following RNA.     

RNA chaperones encoded by RNA viruses

RNAs are functionally diverse macromolecules whose proper functions rely strictly upon their correct tertiary structures. However, because of their high structural flexibility, correct folding of RNAs is challenging and slow. Therefore, cells and viruses encode a variety of RNA remodeling proteins, including helicases and RNA chaperones. In RNA viruses, these proteins are believed to play pivotal roles in all the processes involving viral RNAs during the life cycle. RNA helicases have been studied extensively for decades, whereas RNA chaperones, particularly virus-encoded RNA chaperones, are often overlooked. This review describes the activities of RNA chaperones encoded by RNA viruses, particularly the ones identified and characterized in recent years, and the functions of these proteins in different steps of viral life cycles, and presents an overview of this unique group of proteins.     

Gene amplification and expression by RNA viruses and potential for further application to plant gene transfer

The great majority of plant viruses encapsidate messenger-sense ssRNA and have no natural DNA phase in their life cycle. Despite their RNA nature, essentially any desired change can be introduced into such genomes by using recombinant DNA techniques with suitably constructed, expressible viral cDNA clones. For some viruses such as brome mosaic virus, these methods have been used to define the sequences controlling RNA-directed genomic RNA replication and the expression of internal genes via subgenomic mRNAs. The results suggest a surprising degree of genetic flexibility, which appears to be reflected in the varied gene complements and genetic organizations of presumably related plant and animal RNA viruses sharing conserved replication genes. Foreign genes inserted in such RNA virus genomes can be amplified and expressed to a high level in transfected plant cells. In addition to the potential use of such viruses as episomal expression vectors, it should be possible to couple the viral pathways of RNA-dependent RNA synthesis to amplify and to further regulate the expression of genes transformed into plant chromosomes.     

Reverse genetics of negative-stranded RNA viruses: a global perspective

The advent of reverse genetics technology has revolutionized the field of RNA viruses. It is now possible to manipulate even negative-stranded RNA viruses at will, and evaluate the effects of these changes on the biology and pathogenesis of these viruses. The fundamental insights gleaned from the reverse genetics-based studies over the last several years have provided a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. The recombinant viruses have been exploited also as vectors for devising targeted therapies for non-viral diseases such as malignancies, and in gene therapy for inherited disorders. This review provides a brief summary of the stumbling blocks and the successes in the development of the technology for the negative-stranded RNA viruses. The many and varied applications of the recombinant vectors are also outlined.     

Open reading frame in the minus strand of two plus type RNA viruses

Inspection of the nucleotide sequences of the RNAs complementary to the coat protein mRNAs from two plant viruses with a tripartite genome: alfalfa mosaic virus and brome mosaic virus, showed the presence of open reading frames for 138 and 118 amino acids, respectively. A third virus (cowpea chlorotic mottle virus) from the same family (1) does not show this phenomenon. This suggests that if a protein is coded for by the open reading frames it may be not essential for virus multiplication. Alternatively the open reading frames have no coding function but result from structural requirements of the RNAs.     

RNA viruses as vectors for the expression of heterologous proteins

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.     

Restriction in the cleavage activity of hammerhead ribozymes ensures ongoing evolution in prebiotic RNA world

Self-cleaving infectious RNAs found in many plant viruses and viroids can also cleave intrans and form hammerhead type secondary structure. It has been observed that the cleavage site must contain the triplet GUC. Also, in other cases, the sequence XUY holds good where X = A, C, G, U and Y = A, C, U but not G. The high electronegative nature of guanosine holds the key to its resistance to cleavage which does not allow hybrid formation between the ribozyme and substrate strands. Guanosine resistance to cleavage might have been the starting thrust for the evolution of a translational initiation codon from XUG. A hypothesis is proposed in this regard and its evolutionary consequences are discussed briefly. Presented at the National Symposium on Evolution of Life.     

Incidence and detection of negative-stranded RNA viruses infecting apple and pear trees in California

Novel negative-stranded RNA (nsRNA) viruses have been recently identified in multiple agronomic crops, including pome fruit trees. Citrus concave gum-associated virus (CCGaV), citrus virus A (CiVA) and apple rubbery wood viruses 1 and 2 (ARWV1 and 2) are examples of such viruses. Given the novelty and lack of information about these pathogens in Californian orchards, in this study, real-time RT-PCR assays for CCGaV, CiVA, ARWV1 and 2 were developed and employed in a field survey. Initially, the new assays were challenged against a comprehensive set of positive and negative samples, previously analysed by high-throughput sequencing (HTS), to determine specificity. Aiming to investigate the presence of nsRNA viruses in California apple and pear orchards, 186 samples were collected from 21 different locations. As a result, 79 (42%) samples were found to be infected by these viruses in single or mixed infections. The incidence of each virus in relation to the total number of samples was 36%, 15%, 11% and 0% for ARWV2, CCGaV, ARWV1 and CiVA respectively. Overall, not considering the no detected CiVA, the other three nsRNA viruses were widely distributed among sampled orchards. To further validate the reliability of the new real-time RT-PCR assays, six samples tested positive during the survey were screened by previously described detection assays and HTS. This is the first detection of these nsRNA viruses in California, which may represent an issue in apple and pear production.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

The Nicotinamide Biosynthetic Pathway Is a By-Product of the RNA World

Many of the biosynthetic pathways, especially those leading to the coenzymes, must have originated very early, perhaps before enzymes were available to catalyze their synthesis. While a number of enzymatic reactions in metabolism are known to proceed nonenzymatically, there are no examples of entire metabolic sequences that can be achieved in this manner. The most primitive pathway for nicotinic acid biosynthesis is the reaction of aspartic acid with dihydroxyacetone phosphate. We report here that nicotinic acid (NAc) and its metabolic precursor, quinolinic acid (QA), are produced in yields as high as 7% in a six-step nonenzymatic sequence from aspartic acid and dihydroxyacetone phosphate (DHAP). The biosynthesis of ribose phosphate could have produced DHAP and other three carbon compounds. Aspartic acid could have been available from prebiotic synthesis or from the ribozyme synthesis of pyrimidines. These results suggest that NAD could have originated in the RNA world and that the nonenzymatic biosynthesis of the cofactor nicotinamide could have been an inevitable consequence of life based on carbohydrates and amino acids. The enzymes of the modern pathway were later added in any order. Received: 22 May 2000 / Accepted: 7 August 2000     

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10⁸ molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies—characterized by a fixed number of paired nucleotides—and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited.     

RNA的历史与未来

核酶的发现使得人们有理由相信生命起源于RNA,通过试管演化实验获得的各种各样的催化性RNA更使人们对地球历史早期的RNA世界有了越来越多的了解。同时,随着RNA结构和功能上非凡的多样性的日益被揭示．RNA在未来的临床应用研究中所具有的巨大潜力也正逐渐显现出来。     

Are we missing half of the viruses in the ocean?

Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.     

Omnipotent RNA

The capability of polyribonucleotide chains to form unique, compactly folded structures is considered the basis for diverse non-genetic functions of RNA, including the function of recognition of various ligands and the catalytic function. Together with well-known genetic functions of RNA – coding and complementary replication – this has led to the concept of the functional omnipotence of RNA and the hypothesis that an ancient RNA world supposedly preceded the contemporary DNA–RNA–protein life. It is proposed that the Woese universal precursor in the ancient RNA world could be a cell-free community of mixed RNA colonies growing and multiplying on solid surfaces.     

The RNA World on Ice: A New Scenario for the Emergence of RNA Information

The RNA world hypothesis refers to a hypothetical era prior to coded peptide synthesis, where RNA was the major structural, genetic, and catalytic agent. Though it is a widely accepted scenario, a number of vexing difficulties remain. In this review we focus on a missing link of the RNA world hypothesis—primitive miniribozymes, in particular ligases, and discuss the role of these molecules in the evolution of RNA size and complexity. We argue that prebiotic conditions associated with freezing, rather than “warm and wet” conditions, could have been of key importance in the early RNA world.[Reviewing Editor: Dr. Niles Lehman]