A DNA enzyme that mimics the first step of RNA splicing

We have discovered an artificial DNA enzyme that mimics the first step of RNA splicing. In vitro selection was used to identify DNA enzymes that ligate RNA. One of the new DNA enzymes carries out splicing-related catalysis by specifically recognizing an unpaired internal adenosine and facilitating attack of its 2'-hydroxyl onto a 5'-triphosphate. This reaction forms 2',5'-branched RNA and is analogous to the first step of in vivo RNA splicing, in which a ribozyme cleaves itself with formation of a branched intermediate. Unlike a natural ribozyme, the new DNA enzyme has no 2'-hydroxyl groups to aid in the catalytic mechanism. Our finding has two important implications. First, branch-site adenosine reactivity seems to be mechanistically favored by nucleic acid enzymes. Second, hydroxyl groups are not obligatory components of nucleic acid enzymes that carry out biologically related catalysis.     

A deoxyribozyme that synthesizes 2',5'-branched RNA with any branch-site nucleotide

RNA molecules with internal 2′,5′-branches are intermediates in RNA splicing, and branched RNAs have recently been proposed as retrotransposition intermediates. A broadly applicable in vitro synthetic route to branched RNA that does not require self-splicing introns or spliceosomes would substantially improve our ability to study biochemical processes that involve branched RNA. We recently described 7S11, a deoxyribozyme that was identified by in vitro selection and has general RNA branch-forming ability. However, an important restriction for 7S11 is that the branch-site RNA nucleotide must be a purine (A or G), because a pyrimidine (U or C) is not tolerated. Here, we describe the compact 6CE8 deoxyribozyme (selected using a 20 nt random region) that synthesizes 2′,5′-branched RNA with any nucleotide at the branch site. The Mn²⁺-dependent branch-forming ligation reaction is between an internal branch-site 2′-hydroxyl nucleophile on one RNA substrate with a 5′-triphosphate on another RNA substrate. The preference for the branch-site nucleotide is U > C A > G, although all four nucleotides are tolerated with useful ligation rates. Nearly all other nucleotides elsewhere in both RNA substrates allow ligation activity, except that the sequence requirement for the RNA strand with the 5′-triphosphate is 5′-pppGA, with 5′-pppGAR (R = purine) preferred. These characteristics permit 6CE8 to prepare branched RNAs of immediate practical interest, such as the proposed branched intermediate of Ty1 retrotransposition. Because this branched RNA has two strands with identical sequence that emerge from the branch site, we developed strategies to control which of the two strands bind with the deoxyribozyme during the branch-forming reaction. The ability to synthesize the proposed branched RNA of Ty1 retrotransposition will allow us to explore this important biochemical pathway in greater detail.     

More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns.

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.     

Mimicking the first step of RNA splicing: an artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5'-exon analogue

The 7S11 deoxyribozyme synthesizes 2',5'-branched RNA by mediating the nucleophilic attack of an internal 2'-hydroxyl group of one RNA substrate into the 5'-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5'-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5'-diphosphate or 5'-monophosphate, the same 2',5'-branched product is formed as when pyrophosphate is the leaving group, but with an approximately 50- or approximately 1000-fold lower rate (Br?nsted beta(LG) = -0.40). When the 5'-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5'-terminal alpha-phosphate or the original alpha-phosphate can be attacked by the branch-site 2'-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these alpha-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5'-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5'-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.     

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.     

Control of branch-site choice by a group II intron.

The branch site of group II introns is typically a bulged adenosine near the 3'-end of intron domain 6. The branch site is chosen with extraordinarily high fidelity, even when the adenosine is mutated to other bases or if the typically bulged adenosine is paired. Given these facts, it has been difficult to discern the mechanism by which the proper branch site is chosen. In order to dissect the determinants for branch-point recognition, new mutations were introduced in the vicinity of the branch site and surrounding domains. Single mutations did not alter the high fidelity for proper branch-site selection. However, several combinations of mutations moved the branch site systematically to new positions along the domain 6 stem. Analysis of those mutants, together with a new alignment of domain 5 and domain 6 sequences, reveals a set of structural determinants that appear to govern branch-site selection by group II introns.     

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.     

Statistical properties of the branch-site test of positive selection

The branch-site test is a likelihood ratio test to detect positive selection along prespecified lineages on a phylogeny that affects only a subset of codons in a protein-coding gene, with positive selection indicated by accelerated nonsynonymous substitutions (with ω = d(N)/d(S) > 1). This test may have more power than earlier methods, which average nucleotide substitution rates over sites in the protein and/or over branches on the tree. However, a few recent studies questioned the statistical basis of the test and claimed that the test generated too many false positives. In this paper, we examine the null distribution of the test and conduct a computer simulation to examine the false-positive rate and the power of the test. The results suggest that the asymptotic theory is reliable for typical data sets, and indeed in our simulations, the large-sample null distribution was reliable with as few as 20-50 codons in the alignment. We examined the impact of sequence length, the strength of positive selection, and the proportion of sites under positive selection on the power of the branch-site test. We found that the test was far more powerful in detecting episodic positive selection than branch-based tests, which average substitution rates over all codons in the gene and thus miss the signal when most codons are under strong selective constraint. Recent claims of statistical problems with the branch-site test are due to misinterpretations of simulation results. Our results, as well as previous simulation studies that have demonstrated the robustness of the test, suggest that the branch-site test may be a useful tool for detecting episodic positive selection and for generating biological hypotheses for mutation studies and functional analyses. The test is sensitive to sequence and alignment errors and caution should be exercised concerning its use when data quality is in doubt.     

Branched RNA

The only RNA molecules known to be branched are circular structures with tails known as lariats that arise during nuclear pre-mRNA splicing. Lariats accumulate within a large multicomponent particle called a spliceosome that forms upon the addition of unspliced mRNA to nuclear extracts. Recently an RNA molecule has been observed to catalyze branch formation. In this case a single intron of a yeast mitochondrial pre-mRNA participates in a self-splicing reaction that results in the accumulation of branched lariats that are processed to correctly spliced exons. An enzyme highly specific for branch removal found in the same extracts that form branches during pre-mRNA splicing can debranch RNA lariats to their linear forms without loss of nucleotides. The chemical synthesis of branched RNA has recently been achieved. High yields of sequence-specific oligonucleotides are now available for the analysis of RNA splicing by techniques dependent on branch-site recognition.     

Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase

We have shown previously that CrhC is a unique member of the DEAD-box family of RNA helicases whose expression occurs specifically under conditions of cold stress. Here we show that recombinant His-tagged CrhC, purified from Escherichia coli, is an ATP-independent RNA binding protein possessing RNA-dependent ATPase activity which is stimulated most efficiently by rRNA and polysome preparations. RNA strand displacement assays indicate that CrhC possesses RNA unwinding activity that is adenosine nucleotide specific. Unwinding of partially duplexed RNA proceeds in the 5′→3′ but not the 3′→5′ direction using standard assay conditions. Immunoprecipitation and far-western analysis indicate that CrhC is a component of a multisubunit complex, interacting specifically with a 37 kDa polypeptide. We propose that CrhC unwinds cold-stabilized secondary structure in the 5′-UTR of RNA during cold stress.     

Initial Nucleotide Frequencies of Bacterial RNA synthesized during Amino-acid Starvation or Changes of Carbon Source

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo¹ or in the proper conditions in vitro^2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine⁴. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.     

Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine

Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine residue. The 2' OH of this adenosine, called the branch site, brings about nucleophilic attack at the pre-mRNA 5' splice site in the first step of splicing. Another feature of this pairing is the phylogenetic conservation of a pseudouridine (psi) residue in U2 snRNA nearly opposite the branch site. We show that the presence of this psi in the pre-mRNA branch-site helix of Saccharomyces cerevisiae induces a dramatically altered architectural landscape compared with that of its unmodified counterpart. The psi-induced structure places the nucleophile in an accessible position for the first step of splicing.     

Directing the outcome of deoxyribozyme selections to favor native 3'-5' RNA ligation

Previous experiments have identified numerous RNA ligase deoxyribozymes, each of which can synthesize either 2',5'-branched RNA, linear 2'-5'-linked RNA, or linear 3'-5'-linked RNA. These products may be formed by reaction of a 2'-hydroxyl or 3'-hydroxyl of one RNA substrate with the 5'-triphosphate of a second RNA substrate. Here the inherent propensities for nucleophilic reactivity of specific hydroxyl groups were assessed using RNA substrates related to the natural sequences of spliceosome substrates and group II introns. With the spliceosome substrates, nearly half of the selected deoxyribozymes mediate a ligation reaction involving the natural branch-point adenosine as the nucleophile. In contrast, mostly linear RNA is obtained with the group II intron substrates. Because the two sets of substrates differ at only three nucleotides, we conclude that the location of the newly created ligation junction in DNA-catalyzed branch formation depends sensitively on the RNA substrate sequences. During the experiment that led primarily to branched RNA, we abruptly altered the selection strategy to demand that the deoxyribozymes create linear 3'-5' linkages by introducing an additional selection step involving the 3'-5'-selective 8-17 deoxyribozyme. Although no 3'-5' linkages (相似文献   

RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX.

We used in vitro selection (SELEX) to isolate RNA 'aptamers' to S-adenosyl methionine (SAM). Individual aptamer sequences conform to the structural element noted previously for adenosine binding in selections for aptamers to ATP and NAD+. When we compare the patterns of sequence conservation among 65 adenosine-binding sequences to the published structure of the adenosine aptamer, we find that the most highly conserved nucleotides contact the bound adenosine directly, and that one conserved nucleotide outside the binding pocket is in position to stabilize nucleotides within the binding pocket. The aptamer's ability to bind diverse adenosine-containing cofactors is easily understood in terms of its mode of binding, which leaves the 5'position exposed to solvent. We propose that aptamers that bind their targets away from the reactive moiety may be particularly well suited for catalysis. Finally, we estimate that one sequence in 10(11) may be able to form this structural motif, and that there may be many other adenosine-binding motifs that have escaped detection because of their lower representation in the starting random pools.     

Frequent false detection of positive selection by the likelihood method with branch-site models

Positive Darwinian selection promotes fixations of advantageous mutations during gene evolution and is probably responsible for most adaptations. Detecting positive selection at the DNA sequence level is of substantial interest because such information provides significant insights into possible functional alterations during gene evolution as well as important nucleotide substitutions involved in adaptation. Efficient detection of positive selection, however, has been difficult because selection often operates on only a few sites in a short period of evolutionary time. A likelihood-based method with branch-site models was recently introduced to overcome such difficulties. Here I examine the accuracy of the method using computer simulation. I find that the method detects positive selection in 20%-70% of cases when the DNA sequences are generated by computer simulation under no positive selection. Although the frequency of such false detection varies depending on, among other things, the tree topology, branch length, and selection scheme, the branch-site likelihood method generally gives misleading results. Thus, detection of positive selection by this method alone is unreliable. This unreliability may have resulted from its over-sensitivity to violations of assumptions made in the method, such as certain distributions of selective strength among sites and equal transition/transversion ratios for synonymous and nonsynonymous substitutions.