RNA Biochemistry Research Articles

Structure-Function Relationship of Substituted Bromomethylcoumarins in Nucleoside Specificity of RNA Alkylation

Selective alkylation of RNA nucleotides is an important field of RNA biochemistry, e.g. in applications of fluorescent labeling or in structural probing experiments, yet detailed structure-function studies of labeling agents are rare. Here, bromomethylcoumarins as reactive compounds for fluorescent labeling of RNA are developed as an attractive scaffold on which electronic properties can be modulated by varying the substituents. Six different 4-bromomethyl-coumarins of various substitution patterns were tested for nucleotide specificity of RNA alkylation using tRNA from Escherichia coli as substrate. Using semi-quantitative LC-MS/MS analysis, reactions at mildly acidic and slightly alkaline pH were compared. For all tested compounds, coumarin conjugates with 4-thiouridine, pseudouridine, guanosine, and uridine were identified, with the latter largely dominating. This data set shows that selectivity of ribonucleotide alkylation depends on the substitution pattern of the reactive dye, and even more strongly on the modulation of the reaction conditions. The latter should be therefore carefully optimized when striving to achieve selectivity. Interestingly, the highest selectivity for labeling of a modified nucleoside, namely of 4-thiouridine, was achieved with a compound whose selectivity was somewhat less dependent on reaction conditions than the other compounds. In summary, bromomethylcoumarin derivatives are a highly interesting class of compounds, since their selectivity for 4-thiouridine can be efficiently tuned by variation of substitution pattern and reaction conditions.

Two Doses of Candidate TB Vaccine MVA85A in Antiretroviral Therapy (ART) Naïve Subjects Gives Comparable Immunogenicity to One Dose in ART+ Subjects

Tuberculosis (TB) is a global public health problem exacerbated by the HIV epidemic. Here we evaluate a candidate TB vaccine, MVA85A, in a Phase I study in HIV-infected adults in Senegal. 24 patients were enrolled: Group 1∶12, antiretroviral therapy (ART) naïve, adults, with CD4 counts >300 and HIV RNA load <100 000 copies/ml. Group 2∶12 adults, stable on ART, with CD4 counts >300, and an undetectable HIV RNA load. Safety was evaluated by occurrence of local and systemic adverse events (AEs) and by monitoring of CD4 count, HIV RNA load, haematology and biochemistry. Immunogenicity was evaluated by ex-vivo interferon-gamma ELISpot assay. 87.7% of AEs were mild; 11.6% were moderate; and 0.7% were severe. 29.2% of AEs were systemic; 70.8% were expected local AEs. There were no vaccine-related Serious Adverse Events (SAEs) or clinically significant effects on HIV RNA load or CD4 count. In ART naive subjects, the first MVA85A immunisation induced a significant immune response at 1 and 4 weeks post-immunisation, which contracted to baseline by 12 weeks. Durability of immunogenicity in subjects on ART persisted out to 24 weeks post-vaccination. A second dose of MVA85A at 12 months enhanced immunogenicity in ART naïve subjects. Subjects on ART had higher responses after the first vaccination compared with ART naïve subjects; responses were comparable after 2 immunisations. In conclusion, MVA85A is well-tolerated and immunogenic in HIV-infected subjects in Senegal. A two dose regimen in ART naïve subjects is comparable in immunogenicity to a single dose in subjects on ART.Clinicaltrials.gov trial identifier NCT00731471.

Regulatory RNAs and beyond

The dynamic regulation of biological processes by RNA has emerged as a key field in recent years, and was the topic of the 62nd Mosbacher Colloquium of the German Society for Biochemistry and Molecular Biology (GBM). The 2011 Colloquium, held in April in the romantic Neckar-river region, was also a celebration of the tenth anniversary of the RNA Biochemistry study group within the GBM, which acts as platform for RNA biologists and chemists within Germany and in other European countries.

Discovery of a minimal form of RNase P in Pyrobaculum

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5' leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533-1540]. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5' tRNA leaders. The "missing" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5' pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5' tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.

Structural Stability of Tandemly Occurring Noncanonical Basepairs within Double Helical Fragments: Molecular Dynamics Studies of Functional RNA

Noncanonical basepairs have gained importance over the past few years because of their various functions in RNA biochemistry. These basepairs appear quite frequently in different double helical stems also, but whether they are stabilized by contextual pressure or act as seeds of folding is not yet clear. We have used all-atom molecular dynamics simulations to characterize the stability and functional features of a few noncanonical basepairs within two RNA double helical fragments obtained from ribosome crystal structures. It is anticipated that the noncanonical basepairs would open up spontaneously if they had appeared due to contextual pressure. However, we have found from MD simulations that the noncanonical basepairs occurring in tandem at the central regions of double helical stretches are quite stable. Analysis of basepairing parameters carried out in terms of H-bonding edge-specific axis system indicates that dynamics of the noncanonical basepairs are very similar to those of the canonical ones. The stacking parameters for dinucleotide steps consisting of noncanonical basepairs are rather unusual, but the variability patterns indicate their significant stability. The stacking free-energy values as presumed from the distributions of structural parameters also appear to be similar for both canonical and noncanonical basepair steps.

Preface

Preface

Isothermal titration calorimetry of RNA

Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K a, Δ G, Δ H, Δ S and reaction stoichiometry ( n). Repeating the experiment at different temperatures allows determination of the heat capacity change (Δ C P) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA.

Dissecting RNA folding by nucleotide analog interference mapping (NAIM).

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic approach that allows RNA structure and function to be characterized at the atomic level. Random modifications of base or backbone moieties are incorporated into the RNA transcript as nucleotide analog phosphorothioates. The resulting RNA pool is then subjected to a stringent selection step, in which the RNA has to accomplish a specific task, for example, folding. RNA functional groups important for this process can be identified by physical isolation of the functional and the nonfunctional RNA molecules and subsequent mapping of the modified nucleotide positions in both RNA populations by iodine cleavage of the susceptible phosphorothioate linkage. This approach has been used to analyze a variety of aspects of RNA biochemistry, including RNA structure, catalysis and ligand interaction. Here, I describe how to set up a NAIM assay for studying RNA folding. This protocol can be readily adapted to study any RNAs and their properties. The time required to complete the experiment is dependent on the length of the RNA and the number of atomic modifications tested. In general, a single NAIM experiment can be completed in 1-2 weeks, but expect a time frame of several weeks to obtain reliable and statistically meaningful results.

Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

Human immunodeficiency virus type 1 minus strand transfer was measured using a genomic donor-acceptor template system in vitro. Donor RNA D199, having the minimum region required for minus strong stop DNA synthesis, was previously shown to transfer with 35% efficiency to an acceptor RNA representing the 3' repeat region. Donor D520, having an additional 321-nucleotide segment extending into gag, transferred at 75% efficiency. In this study each transfer step was analyzed to account for the difference. Measurement of terminal transfer indicated that the 3' terminus of the cDNA generated using D520 is more accessible for transfer than that of D199. Nevertheless, acceptor competition experiments demonstrated that D520 has a greater preference for invasion-driven versus terminal transfer than D199. Competition mapping showed that the base of the transactivation response element is the primary invasion site for D520, important for efficient acceptor invasion. Acceptors complementary to the invasion and terminal transfer sites, but not the region between, allowed assessment of the significance of hybrid propagation by branch migration. These bipartite acceptors showed that with D520, invasion raises the local concentration of the acceptor for efficient terminal transfer by a proximity effect. However, with D199, invasion is relatively inefficient, and the cDNA 3' terminus is not very accessible. For most transfers that occurred, the acceptor accessed the cDNA 3' end by branch migration. Results suggest that both proximity and branch migration mechanisms contribute to transfers, with the proportion determined by donor-cDNA structure. D520 transfers better because it has greater accessibility for both invasion and terminus transfer.

RNA 1997–2007: A Remarkable Decade of Discovery

The past decade has witnessed spectacular progress in the field of RNA biology and biochemistry; old problems have been solved, and new ones have emerged. This perspective briefly reviews where we are today and where we might be 10 years hence.

Transcription Termination Factor Rho Can Displace Streptavidin from Biotinylated RNA

In Escherichia coli, binding of the hexameric Rho protein to naked C-rich Rut (Rho utilization) regions of nascent RNA transcripts initiates Rho-dependent termination of transcription. Although the ring-shaped Rho factor exhibits in vitro RNA-dependent ATPase and directional RNA-DNA helicase activities, the actual molecular mechanisms used by Rho to disrupt the intricate network of interactions that cement the ternary transcription complex remain elusive. Here, we show that Rho is a molecular motor that can apply significant disruptive forces on heterologous nucleoprotein assemblies such as streptavidin bound to biotinylated RNA molecules. ATP-dependent disruption of the biotin-streptavidin interaction demonstrates that Rho is not mechanistically limited to the melting of nucleic acid base pairs within molecular complexes and confirms that specific interactions with the roadblock target are not required for Rho to operate properly. We also show that Rho-induced streptavidin displacement depends significantly on the identity of the biotinylated transcript as well as on the position, nature, and length of the biotin link to the RNA chain. Altogether, our data are consistent with a "snow plough" type of mechanism of action whereby an early rearrangement of the Rho-substrate complex (activation) is rate-limiting, physical force (pulling) is exerted on the RNA chain by residues of the central Rho channel, and removal of structural obstacles from the RNA track stems from their nonspecific steric exclusion from the hexamer central hole. In this context, a simple model for the regulation of Rho-dependent termination based on the modulation of disruptive dynamic loading by secondary factors is proposed.

Applications of isothermal titration calorimetry in RNA biochemistry and biophysics

Isothermal titration calorimetry (ITC) has been applied to the study of proteins for many years. Its use in the biophysical analysis of RNAs has lagged significantly behind its use in protein biochemistry, however, in part because of the relatively large samples required. As the instrumentation has become more sensitive, the ability to obtain high quality data on RNA folding and RNA ligand interactions has improved dramatically. This review provides an overview of the ITC experiment and describes recent work on RNA systems that have taken advantage of its versatility for the study of small molecule binding, protein binding, and the analysis of RNA folding.

Interactions of the RNA Polymerase with the Viral Genome at the 5′- and 3′-Ends Contribute to 20S RNA Narnavirus Persistence in Yeast

20S RNA narnavirus is a positive strand RNA virus found in the yeast Saccharomyces cerevisiae. The viral genome (2514 nucleotides) only encodes a single protein (p91), the RNA-dependent RNA polymerase and does not have capsid proteins to form intracellular virions. The genomic RNA has no 3' poly(A) tail and perhaps no cap structure at the 5'-end; thus resembling an intermediate of mRNA degradation. The virus, however, escapes the host surveillance and replicates in the yeast cytoplasm persistently. The viral genome is not naked but exists in the form of a ribonucleoprotein complex with p91 in a 1:1 stoichiometry. Here we investigated interactions between p91 and the viral genome. Our results indicate that p91 directly or indirectly interacts with the RNA at the 5'- and 3'-end regions and to a lesser extent at a central part. The 3'-end site is identical to or overlaps with the 3' cis signal for replication identified previously. The 5'-site is at the second stem loop structure from the 5'-end (nucleotides 72-104), and this structure also contains a cis signal for replication. Analysis of mutants in the structure revealed a tight correlation between replication and formation of complexes. These results highlight the importance of ribonucleoprotein complexes for the viral life cycle. We will discuss implications of these findings especially on how the virus escapes from mRNA degradation pathways and resides in the cytoplasm persistently despite the lack of a protective capsid.

Group II introns: structure, folding and splicing mechanism

Group II introns are large autocatalytic RNAs found in organellar genomes of plants and lower eukaryotes, as well as in some bacterial genomes. Interestingly, these ribozymes share characteristic traits with both spliceosomal introns and non-LTR retrotransposons and may have a common evolutionary ancestor. Furthermore, group II intron features such as structure, folding and catalytic mechanism differ considerably from those of other large ribozymes, making group II introns an attractive model system to gain novel insights into RNA biology and biochemistry. This review explores recent advances in the structural and mechanistic characterization of group II intron architecture and self-splicing.

RNA world – the dark matter of evolutionary genomics

For a long time, molecular evolutionary biologists have been focused on DNA and proteins, whereas RNA has lived in the shadow of its famous chemical cousins as a mere intermediary. Although this perspective has begun to change since genome-wide transcriptional profiling was successfully extended to evolutionary biology, it still echoes in evolutionary literature. In this mini-review, new developments of RNA biochemistry and transcriptomics are brought to the attention of evolutionary biologists. In particular, the unexpected abundance and functional significance of noncoding RNAs is briefly reviewed. Noncoding RNAs control a remarkable range of biological pathways and processes, all with obvious fitness consequences, such as initiation of translation, mRNA abundance, transposon jumping, chromosome architecture, stem cell maintenance, development of brain and muscles, insulin secretion, cancerogenesis and plant resistance to viral infections.

In vitro Transcription and Purification of RNAs of Different Size

In vitro run-off transcription from a double-stranded DNA template by T7 RNA polymerase is an elegant way to obtain highly pure and uniform RNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides. Here we describe the different strategies applied and optimized in our laboratory to enzymatically synthesize RNAs as necessary when working at the interface of bioinorganic chemistry, coordination chemistry, RNA biochemistry and structural biology.

In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg2+ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.

A More Complex Isoleucine Aptamer with a Cognate Triplet

The simplest RNA that can meet a column affinity selection for isoleucine was previously defined using selection amplification with decreasing numbers of randomized nucleotides. This simplest UAUU motif was a small asymmetric internal loop. Conserved positions of the loop include isoleucine codon and anticodon triplets (Lozupone C., Changayil, S., Majerfeld, I., and Yarus, M. (2003) RNA (N. Y.) 9, 1315-1322). Using new primer sequences, we now select a somewhat more complex isoleucine binding RNA, requiring 4.7 more bits of information to describe. The newly selected structure is a terminal or hairpin loop of 20 nucleotides, 15 being invariant. An information profile shows that the new binding site contains five short functional loop regions joined by less significant single nucleotide positions. Among the important nucleotides is a conserved isoleucine anticodon, supporting the escaped triplet theory, which posits a stereochemical genetic code originating in RNA amino acid binding sites.

Leon Heppel and the Early Days of RNA Biochemistry

Today, machines turn out the sequence of a million DNA bases in a day. Fifty years ago, when the chemical and biochemical tools for studying DNA and RNA were at best rudimentary, such a machine was unimaginable. Then, the cutting edge was Erwin Chargaff's demonstration, in 1948, that the base composition of DNA could be reliably determined. His discovery that all DNAs contain equal amounts of adenine and thymine and similarly of guanine and cytosine depended on applying two recent developments: partition chromatography and the absorption spectra of nucleic acid constituents.

Editorial: RNA–ligand interactions

Editorial: RNA–ligand interactions