Chemical synthesis of branched RNA.

A branched tetranucleotide consisting of adenosine linked 2' and 5' to guanosine and 3' to cytidine was synthesized from appropriately protected nucleoside phosphoramidites as synthons. The product was characterized enzymatically.     

Characterization of deoxyribozymes that synthesize branched RNA

We recently reported deoxyribozymes (DNA enzymes) that synthesize 2',5'-branched RNA. The in vitro-selected 9F7 and 9F21 deoxyribozymes mediate reaction of a branch-site adenosine 2'-hydroxyl on one RNA substrate with the 5'-triphosphate of another RNA substrate. Here we characterize these DNA enzymes with respect to their branch-forming activity. Both 9F7 and 9F21 are much more active with Mn(2+) than with Mg(2+). The K(d,app)(Mg(2+)) > 400 mM but K(d,app)(Mn(2+)) approximately 20-50 mM, and the ligation rates k(obs) are orders of magnitude faster with Mn(2+) than with Mg(2+) (e.g., 9F7 approximately 0.3 min(-1) with 20 mM Mn(2+) versus 0.4 h(-1) with 100 mM Mg(2+), both at pH 7.5 and 37 degrees C). Of the other tested transition metal ions Zn(2+), Ni(2+), Co(2+), and Cd(2+), only Co(2+) supports a trace amount of activity. 9F7 is more tolerant than 9F21 of varying the RNA substrate sequences. For the RNA substrate that donates the adenosine 2'-hydroxyl, 9F7 requires YUA, where Y = pyrimidine and A is the branch site. The 3'-tail emerging from the branch-site A may have indefinite length, but it must be at least one nucleotide long for high activity. The 5'-triphosphate RNA substrate requires several additional nucleotides with varying sequence requirements (5'-pppGRMWR). Outside of these regions that flank the ligation site, 9F7 and 9F21 tolerate any RNA substrate sequences via Watson-Crick covariation of the DNA binding arms that interact directly with the substrates. 9F7 provides a high yield of 2',5'-branched RNA on the preparative nanomole scale. The ligation reaction is effectively irreversible; the pyrophosphate leaving group in the ligation reaction does not induce 2',5'-cleavage, and pyrophosphate does not significantly inhibit ligation except in 1000-fold excess. Deleting a specific nucleotide in one of the DNA binding arms near the ligation junction enhances ligation activity, suggesting an interesting structure near this region of the deoxyribozyme-substrate complex. These data support the utility of deoxyribozymes in creating synthetic 2',5'-branched RNAs for investigations of group II intron splicing, debranching enzyme (Dbr) activity, and other biochemical reactions.     

Salt-Dependent Folding Energy Landscape of RNA Three-Way Junction

RNAs are highly negatively charged chain molecules. Metal ions play a crucial role in RNA folding stability and conformational changes. In this work, we employ the recently developed tightly bound ion (TBI) model, which accounts for the correlation between ions and the fluctuation of ion distributions, to investigate the ion-dependent free energy landscape for the three-way RNA junction in a 16S rRNA domain. The predicted electrostatic free energy landscape suggests that 1), ion-mediated electrostatic interactions cause an ensemble of unfolded conformations narrowly populated around the maximally extended structure; and 2), Mg²⁺ ion-induced correlation effects help bring the helices to the folded state. Nonelectrostatic interactions, such as noncanonical interactions within the junctions and between junctions and helix stems, might further limit the conformational diversity of the unfolded state, resulting in a more ordered unfolded state than the one predicted from the electrostatic effect. Moreover, the folded state is predominantly stabilized by the coaxial stacking force. The TBI-predicted folding stability agrees well with the experimental measurements for the different Na⁺ and Mg²⁺ ion concentrations. For Mg²⁺ solutions, the TBI model, which accounts for the Mg²⁺ ion correlation effect, gives more improved predictions than the Poisson-Boltzmann theory, which tends to underestimate the role of Mg²⁺ in stabilizing the folded structure. Detailed control tests indicate that the dominant ion correlation effect comes from the charge-charge Coulombic correlation rather than the size (excluded volume) correlation between the ions. Furthermore, the model gives quantitative predictions for the ion size effect in the folding energy landscape and folding cooperativity.     

RNA Folding Argues Against a Hot-Start Origin of Life

Opinion is strongly divided on whether life arose on earth under hot or cold conditions, the hot-start and cold-start scenarios, respectively. The origin of life close to deep thermal vents appears as the majority opinion among biologists, but there is considerable biochemical evidence that high temperatures are incompatible with an RNA world. To be functional, RNA has to fold into a three-dimensional structure. We report both theoretical and experimental results on RNA folding and show that (as expected) hot conditions strongly reduce RNA folding. The theoretical results come from energy-minimization calculations of the average extent of folding of RNA, mainly from 0–90°C, for both random sequences and tRNA sequences. The experimental results are from circular-dichroism measurements of tRNA over a similar range of temperatures. The quantitative agreement between calculations and experiment is remarkable, even to the shape of the curves indicating the cooperative nature of RNA folding and unfolding. These results provide additional evidence for a lower temperature stage being necessary in the origin of life. Received: 1 March 2000 / Accepted: 14 June 2000     

Conformational properties of branched RNA fragments in aqueous solution

The conformational properties of branched trinucleoside diphosphates ACC, ACG, AGC, AGG, AUU, AGU, AUG, ATT, GUU, and aAUU [XYZ = X(2'p5'Y)3'p5'Z] have been studied in aqueous solution by nuclear magnetic resonance (1H, 13C), ultraviolet absorption, and circular dichroism. It is concluded from these studies that the purine ring of the central residue (X; e.g., adenosine) forms a base-base stack exclusively with the purine or pyrimidine ring of the 2'-nucleotidyl unit (Y; 2'-residue). The residue attached to the central nucleoside via the 3'-5'-linkage (Z; 3'-residue) is "free" from the influence of the other two heterocyclic rings. The ribose rings of the central nucleoside and the 2'- and 3'-residues exist as equilibrium mixtures of C2'-endo (2E)-C3'-endo (3E) conformers. The furanose ring of the central nucleoside (e.g., A) when linked to a pyrimidine nucleoside via the 2'-5'-linkage shows a higher preference for the 2E pucker conformation (e.g., AUG, AUU, ACG, ca. 80%) than those linked to a guanosine nucleoside through the same type of bond (AGU, AGG, AGC, ca. 70%). This indicates some correlation between nucleotide sequence and ribose conformational equilibrium. The 2E-3E equilibrium of 2'-pyrimidines (Y) shows significant, sometimes exclusive, preference (70-100%) for the 3E conformation; 3'-pyrimidines and 2'-guanosines have nearly equal 2E and 3E rotamer populations; and the ribose conformational equilibrium of 3'-guanosines shows a preference (60-65%) for the 2E pucker. Conformational properties were quantitatively evaluated for most of the bonds (C4'-C5', C5'-O5', C2'-O2', and C3'-O3') in the branched "trinucleotides" AUU and AGG by analysis of 1H-1H, 1H-31P, and 13C-31P coupling constants. The C4'-C5' bond of the adenosine units shows a significant preference for the gamma + conformation. The dominant conformation about C4'-C5' and C5'-O5' for the 2'-and 3'-nucleotidyl units is gamma + and beta t, respectively, with larger gamma + and beta t rotamer populations for the 2'-unit. The increased conformational purity in the 2'-residue, compared to the 3'-residue, is ascribed to the presence of an ordered (adenine----2'-residue) stacked state. The favored rotamers about C3'-O3' and C2'-O2' are epsilon- and epsilon'-, respectively. The conformational features of AUU and AGG were compared to those of their constitutive dimers A3'p5'G, A2'p5'G, A3'p5'U, and A2'p5'U and monomers 5'pG and 5'pU.     

Folding kinetics of the lipoic acid-bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex

Intracellular calcium is a second messenger involved in several processes in yeast, such as mating, nutrient sensing, stress response and cell cycle events. It was reported that glucose addition stimulates a rapid increase in free calcium level in yeast. To investigate the calcium level variations induced by different stimuli we used a reporter system based on the photoprotein aequorin. Glucose addition (50 mM) to nutrient-starved cells induced an increase in free intracellular calcium concentration, mainly due to an influx from external medium. The increase of calcium reached its maximum 100–120 s after the stimulus. A concentration of about 20 mM glucose was required for a 50% increase in intracellular calcium. This response was completely abolished in strain plc1Δ and in the isogenic wild-type strain treated with 3-nitrocoumarin, a phosphatidylinositol-specific phospholipase C inhibitor, suggesting that Plc1p is essential for glucose-induced calcium increase. This suggests that Plc1p should have a significant role in transducing glucose signal. The calcium influx induced by addition of high glucose on cells previously stimulated with low glucose levels was inhibited in strains with a deletion in the GPR1 or GPA2 genes, which suggests that glucose would be detected through the Gpr1p/Gpa2p receptor/G protein-coupled (GPCR) complex. Moreover, the signal was completely abolished in a strain unable to phosphorylate glucose, which is consistent with the reported requirement of glucose phosphorylation for GPCR complex activation.     

基于动态权重匹配的RNA折叠算法

本文在最大权重匹配（Maximum Weighted Matching,MWM）算法的基顾础上引入与茎区长度相关的动态权重,采用一种递归算法逐步寻找具有最大权重和的茎区。从而最终确定RNA的二级结构．该算法避开了繁杂的自由能计算,同样也能达到较高的预测精确度并且还能预测到大多数类型的潜在假结（pseudoknots）．     

Hidden Structural Modules in a Cooperative RNA Folding Transition

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A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudo-metallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5′-exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.     

A branched pathway for transgene-induced RNA silencing in plants

In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS). Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene and with methylation of the coding sequence. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ( this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.     

Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn²⁺ as a cofactor, rather than Mg²⁺ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has k_obs on the order of 0.1 min⁻¹, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.     

Folding of a universal ribozyme: the ribonuclease P RNA

Ribonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.     

The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription

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RNA editing in hepatitis delta virus genotype III requires a branched double-hairpin RNA structure

RNA editing at the amber/W site plays a central role in the replication scheme of hepatitis delta virus (HDV), allowing the virus to produce two functionally distinct forms of the sole viral protein, hepatitis delta antigen (HDAg), from the same open reading frame. Editing is carried out by a cellular activity known as ADAR (adenosine deaminase), which acts on RNA substrates that are at least partially double stranded. In HDV genotype I, editing requires a highly conserved base-paired structure that occurs within the context of the unbranched rod structure characteristic of HDV RNA. This base-paired structure is disrupted in the unbranched rod of HDV genotype III, which is the most distantly related of the three known HDV genotypes and is associated with the most severe disease. Here I show that RNA editing in HDV genotype III requires a branched double-hairpin structure that deviates substantially from the unbranched rod structure, involving the rearrangement of nearly 80 bp. The structure includes a UNCG RNA tetraloop, a highly stable structural motif frequently involved in the folding of large RNAs such as rRNA. The double-hairpin structure is required for editing, and hence for virion formation, but not for HDV RNA replication, which requires the unbranched rod structure. HDV genotype III thus relies on a dynamic conformational switch between the two different RNA structures: the unbranched rod characteristic of HDV RNA and a branched double-hairpin structure that is required for RNA editing. The different mechanisms of editing in genotypes I and III underscore their functional differences and may be related to pathogenic differences as well.