Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation

The 5' ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3' end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3' end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed.     

Relics from the RNA World

An RNA world is widely accepted as a probable stage in the early evolution of life. Two implications are that proteins have gradually replaced RNA as the main biological catalysts and that RNA has not taken on any major de novo catalytic function after the evolution of protein synthesis, that is, there is an essentially irreversible series of steps RNA → RNP → protein. This transition, as expected from a consideration of catalytic perfection, is essentially complete for reactions when the substrates are small molecules. Based on these principles we derive criteria for identifying RNAs in modern organisms that are relics from the RNA world and then examine the function and phylogenetic distribution of RNA for such remnants of the RNA world. This allows an estimate of the minimum complexity of the last ribo-organism—the stage just preceding the advent of genetically encoded protein synthesis. Despite the constraints placed on its size by a low fidelity of replication (the Eigen limit), we conclude that the genome of this organism reached a considerable level of complexity that included several RNA-processing steps. It would include a large protoribosome with many smaller RNAs involved in its assembly, pre-tRNAs and tRNA processing, an ability for recombination of RNA, some RNA editing, an ability to copy to the end of each RNA strand, and some transport functions. It is harder to recognize specific metabolic reactions that must have existed but synthetic and bio-energetic functions would be necessary. Overall, this requires that such an organism maintained a multiple copy, double-stranded linear RNA genome capable of recombination and splicing. The genome was most likely fragmented, allowing each ``chromosome' to be replicated with minimum error, that is, within the Eigen limit. The model as developed serves as an outgroup to root the tree of life and is an alternative to using sequence data for inferring properties of the earliest cells. Received: 14 January 1997 / Accepted: 19 May 1997     

Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme

ABSTRACT: BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65 deg C should reduce the constraints of RNA secondary structure in RNA ligation experiments.     

Kinetic and thermodynamic characterization of the RNA guanylyltransferase reaction

An RNA guanylyltransferase activity is involved in the synthesis of the cap structure found at the 5' end of eukaryotic mRNAs. The RNA guanylyltransferase activity is a two-step ping-pong reaction in which the enzyme first reacts with GTP to produce the enzyme-GMP covalent intermediate with the concomitant release of pyrophosphate. In the second step of the reaction, the GMP moiety is then transferred to a diphosphorylated RNA. Both reactions were previously shown to be reversible. In this study, we report a biochemical and thermodynamic characterization of both steps of the reaction of the RNA guanylyltransferase from Paramecium bursaria Chlorella virus 1, the prototype of a family of viruses infecting green algae. Using a combination of real-time fluorescence spectroscopy, radioactive kinetic assays, and inhibition assays, the complete kinetic parameters of the RNA guanylyltransferase were determined. We produced a thermodynamic scheme for the progress of the reaction as a function of the energies involved in each step. We were able to demonstrate that the second step comprises the limiting steps for both the direct and reverse overall reactions. In both cases, the binding to the RNA substrates is the step requiring the highest energy and generating unstable intermediates that will promote the catalytic activites of the enzyme. This study reports the first thorough kinetic and thermodynamic characterization of the reaction catalyzed by an RNA capping enzyme.     

Rearrangements within human spliceosomes captured after exon ligation

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5′ and 3′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.     

Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2'-O-methylation of the 3'-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.     

Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells

Background

RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers.

Results

To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5’ pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors.

Conclusions

Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65°C should reduce the constraints of RNA secondary structure in RNA ligation experiments.     

Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.     

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.     

Demonstration of a multistep mechanism for assembly of the SRP x SRP receptor complex: implications for the catalytic role of SRP RNA

Two GTPases in the signal recognition particle (SRP) and its receptor (SR) control the delivery of newly synthesized proteins to the endoplasmic reticulum or plasma membrane. During the protein targeting reaction, the 4.5S SRP RNA accelerates the association between the two GTPases by 400-fold. Using fluorescence resonance energy transfer, we demonstrate here that formation of a stable SRP·SR complex involves two distinct steps: a fast initial association between SRP and SR to form a GTP-independent early complex and then a GTP-dependent conformational rearrangement to form the stable final complex. We also found that the 4.5S SRP RNA significantly stabilizes the early GTP-independent intermediate. Furthermore, mutational analyses show that there is a strong correlation between the ability of the mutant SRP RNAs to stabilize the early intermediate and their ability to accelerate SRP·SR complex formation. We propose that the SRP RNA, by stabilizing the early intermediate, can give this transient intermediate a longer life time and therefore a higher probability to rearrange to the stable final complex. This provides a coherent model that explains how the 4.5S RNA exerts its catalytic role in SRP·SR complex assembly.     

Differences in the effects of manganese and magnesium on initiation and elongation in the RNA polymerase I reaction

The effects of magnesium and manganese in the initiation and elongation steps of the RNA polymerase I reaction in RNA synthesis were studied. For RNA chain initiation manganese was found to be a better effector than magnesium. For RNA chain elongation either manganese or magnesium acted as an effector, but a high concentration of manganese was inhibitory.     

Antigens Associated with Ribonucleic Acid

The ability of RNA to elicit the formation of certain antibodies in rabbits was investigated by means of the ring test and the complement fixation reaction. As a result, it was found that crude RNA treated with phenol and pure RNA obtained from the phenol-treated RNA had a sensitizing ability. However, this ability was weak and the antibody elicited was a non-precipitating, incomplete one.     

Inhibition of the initiation of leukemic transcription by N-trifluoroacetyladriamycin-14-O-hemiadipate in vitro. Impaired formation of RNA polymerase-DNA complex

The effect of N-trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a new derivative of adriamycin, on various steps of the enzymic reaction catalyzed by chicken myeloblastosis RNA polymerase II was studied. AD 143 inhibition of RNA synthesis, which was evident at the beginning of the reaction, could not be reversed by increasing the concentrations of any one of the four nucleoside triphosphate substrates of the reaction. Furthermore, the RNA synthesis inhibition was not affected by varying the concentrations of template DNA. The AD 143-induced inhibition caused a reduction of the frequency of RNA chain initiation, whereas the average chain length of RNA synthesized at the end of the reaction remained unaltered. The susceptible step in the initiation process was found to be the formation of stable complexes between RNA polymerase and the DNA template. While AD 143 causes no inhibition of Escherichia coli RNA polymerase activity, it was found not to affect the E. coli RNA polymerase-template DNA complex formation.     

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.