An axial binding site in the Tetrahymena precursor RNA.

Previous studies allow the construction of three distinct models of the binding of G and arginine within the active site of the Tetrahymena self-splicing preribosomal precursor RNA. These models (base triple, axial I and axial II) are now distinguished by measurements on the specificity of RNAs with nucleotide substitutions at positions spanning the site. Because the semi-conserved unpaired nucleotide 263 has no effect on substrate or inhibitor selection by the Tetrahymena RNA we conclude that the axial I model is improbable. In contrast, data with substituted RNAs and nucleoside analogs suggest that nucleotide 265 makes a hydrogen bond with the substrate. Accordingly the active site appears axial because substrate contacts exist at more than one nucleotide on the 5' side of the P7 helix. The effects of this hydrogen bond are observable in cases where the donor or acceptor is on the RNA, whether nucleotide 265 is a purine or pyrimidine, or whether nucleotide 265 is mispaired, wobble paired or normally paired. This pattern is consistent with the axial II model. Molecular dynamics and energy minimization calculations lead to the same conclusions as these site-directed substitutions; the base triple and axial I models are unstable dynamically. Under thermal agitation, the third model site (axial II) is transformed to a related, but more stable structure, axial III. The axial III active site is characterized by the extrusion of the conserved bulged base 263 from the P7 helix, a semi-pocket for G base formed by stacking of nucleotide 262, and formation of all bonds to the G base originally proposed for both the base triple and axial II sites. Because of these hydrogen bonds the axial III site is also consistent with data on enzymatic specificity. The axial III model indicates an unforeseen capacity for pocket formation within the groove of an RNA helix, suggests that the site may be unusually flexible, and bears on a hypothesis concerning the origin of the genetic code.     

Specific arginine mediated RNA recognition

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.     

An RNA-amino acid complex and the origin of the genetic code

The group I RNAs, of which the Tetrahymena ribosomal RNA intron is the most investigated example, catalyze their own splicing reactions. Splicing is initiated at a conserved site on the RNA that facilitates attack by exogenous guanosine (or its nucleotides) on the exon-intron junction. The guanosine site in the RNA's catalytic center also binds arginine, and is quite selective for the arginine side chain. This amino acid-RNA interaction is stereoselective, and L-arginine is preferred. Immediately at the site at which arginine binds there is one of only four RNA triplets in 92 group I RNA sequences: AGA/G and CGA/G. Thus the arginine contact site is within any of four different codons for arginine. Mutation of the conserved G in the middle of the triplet decreases affinity for the amino acid, showing that binding is sequence-specific. A pathway for the origin of the genetic code for arginine is suggested, based on the existence and properties of this sequence-specific, amino acid-specific RNA complex. The existence of a proto-ribosome related to the group I RNAs seems the most likely hypothesis. This notion is used to distinguish three periods in the development of the code. Restrained and exuberant hypotheses about the origin of the genetic code are distinguished, and some objections to these hypotheses are considered.     

Selection of small molecules by the Tetrahymena catalytic center.

The catalytic center in group I RNAs contains a selective binding site that accommodates both guanosine and L-arginine. In order to understand the specificity of the RNA for small molecules, we analyzed 6 RNAs that vary in this region. Specificity for nucleotides resides substantially in G264 rather than its paired nucleotide C311, and is expressed substantially in Km, with comparatively little variation in kcat. kcat is not notably perturbed even for RNAs with mispairs in the active-site helix. For 5 of 6 sequences, effects of RNA substitutions on arginine binding and GTP reactivity are proportional, confirming that arginine contacts a subset of the groups occupied by G. As a result of particular mutations, reaction with GTP is decreased, and reaction with the natural nucleotides UTP and ATP is enhanced. Molecular modeling of these effects suggests that exceptionally flexible placement of reactants may be an essential quality of RNA-catalyzed splicing. The specificity of the intron can be rationalized by a type of binding model not previously considered, in which the G/arginine site includes adjacent nucleotides (an 'axial' site), rather than a single nucleotide, G264.     

Requirements for cleavage by a modified RNase P of a small model substrate.

M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, has been covalently linked at its 3' terminus to oligonucleotides (guide sequences) that guide the enzyme to target RNAs through hybridization with the target sequences. These constructs (M1GS RNAs) have been used to determine some minimal features of model substrates. As few as 3 bp on the 3' side of the site of cleavage in a substrate complex and 1 nt on the 5' side are required for cleavage to occur. The cytosines in the 3' terminal CCA sequence of the model substrates are important for cleavage efficiency but not cleavage site selection. A purine (base-paired or not) at the 3' side of the cleavage site is important both for cleavage site selection and efficiency. M1GS RNAs provide both a simple system for characterization of the reaction governed by M1 RNA and a tool for gene therapy.     

Experimental identification and analysis of macronuclear non-coding RNAs from the ciliate Tetrahymena thermophila

The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40-500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena-specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5'-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena. Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.     

Optimizing the substrate specificity of a group I intron ribozyme

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.     

Conversion of mammalian tRNA 3' processing endoribonuclease to four-base-recognizing RNA cutters.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) activities in mammalian cell extracts require both protein and 3' truncated tRNA, species of which direct their substrate sequence specificity. Computer analysis for searching possible base pairing between substrate RNAs and their corresponding 3' truncated tRNA, suggested a unified model for substrate recognition mechanism, in which a four-nucleotide (nt) sequence in the target tRNAs 1 nt upstream of their cleavage site, base pairs with the 5' terminal 4 nt sequence of their corresponding 3' truncated tRNA. This model was supported by experiments with several RNA substrates containing a substituted nucleotide in the target 4 nt sequence. In this model, the tRNA substrates and their corresponding 3' truncated tRNA form a complex resembling a 5' processed tRNA precursor containing a 3' trailer, suggesting that the protein component of RNase 65 is identical to tRNA 3' processing endoribonuclease (3' tRNase). Actually, 3' tRNase purified from pig liver cleaved the target RNAs at the expected sites only in the presence of their corresponding 3' truncated tRNA. These results show that the 3' tRNase can be converted to 4 nt specific RNA cutters using the 3' truncated tRNAs.     

Docking simulation with a purine nucleoside specific homology model of deoxycytidine kinase, a target enzyme for anticancer and antiviral therapy

5'-Phosphorylation, catalyzed by human deoxycytidine kinase (dCK), is a crucial step in the metabolic activation of anticancer and antiviral nucleoside antimetabolites, such as cytarabine (AraC), gemcitabine, cladribine (CdA), and lamivudine. Recently, crystal structures of dCK (dCKc) with various pyrimidine nucleosides as substrates have been reported. However, there is no crystal structure of dCK with a bound purine nucleoside, although purines are good substrates for dCK. We have developed a model of dCK (dCKm) specific for purine nucleosides based on the crystal structure of purine nucleoside bound deoxyguanosine kinase (dGKc) as the template. dCKm is essential for computer aided molecular design (CAMD) of novel anticancer and antiviral drugs that are based on purine nucleosides since these did not bind to dCKc in our docking experiments. The active site of dCKm was larger than that of dCKc and the amino acid (aa) residues of dCKm and dCKc, in particular Y86, Q97, D133, R104, R128, and E197, were not in identical positions. Comparative docking simulations of deoxycytidine (dC), cytidine (Cyd), AraC, CdA, deoxyadenosine (dA), and deoxyguanosine (dG) with dCKm and dCKc were carried out using the FlexX docking program. Only dC (pyrimidine nucleoside) docked into the active site of dCKc but not the purine nucleosides dG and dA. As expected, the active site of dCKm appeared to be more adapted to bind purine nucleosides than the pyrimidine nucleosides. While water molecules were essential for docking experiments using dCKc, the absence of water molecules in dCKm did not affect the ability to correctly dock various purine nucleosides.     

Trans splicing of mRNA precursors in vitro

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.     

Specificity of arginine binding by the Tetrahymena intron

L-Arginine competitively inhibits the reaction of GTP with the Tetrahymena ribosomal self-splicing intron. In order to define this RNA binding site for arginine, Ki's have now been measured for numerous arginine-like competitive inhibitors. Detailed consideration of the Ki's suggests a tripartite binding model. The dissociation constants of the inhibitors can be consistently interpreted if the guanidino group of arginine binds in the GTP site by utilizing the H-bonds otherwise made to the N1-H and 2 NH2 of the guanine pyrimidine ring. The positive charge of the arginine guanidino group also enhances binding. A second requirement is for the precise length of the aliphatic arm connecting the guanidino with the alpha-carbon. The positive charge of the alpha-amino group is the third feature essential to effective inhibition. The negative carboxyl charge of arginine inhibits binding, and the substituents on the alpha-carbon are probably oriented, with the alpha-amino group near the phosphate backbone of the RNA. This orientation contributes strongly to the L stereoselectivity of the amino acid site on the RNA. When spaced optimally, net contribution to the free energy of binding is of the same order for the guanidino group and for the arginine alpha-carbon substituents, but the guanidino apparently contributes more to binding free energy. Taken together, these observations extend the previous binding model [Yarus, M. (1988) Science (Washington, D.C.) 240, 1751-1758]. The observed dependence of binding on universal characteristics of amino acids suggests that RNA binding sites with other amino acid specificities could exist.     

Estimation of whole-body RNA catabolism based on the urinary modified nucleoside levels of cancer and AIDS patients.

From the relationship between the molar ratio of nucleosides calculated stoichiometrically from modified nucleoside occurrences in major RNA species and the proportion of rRNA to all of RNA contents in average tissues, the increase of rRNA contents in cancer tissues growing rapidly was found. Thus, we found that selected urinary modified nucleoside levels were very useful as a biological marker of cancer and AIDS, as well as a good indicator of whole-body metabolic conditions of RNAs.     

Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase.

The in vitro fidelity of the virion-associated RNA polymerase of vesicular stomatitis virus was quantitated for a single conserved viral RNA site and the usual high in vitro base misincorporation error frequencies (approx. 10(-3)) were observed at this (guanine) site. We sought evidence for RNA 3'-->5' exonuclease proofreading mechanisms by varying the concentrations of the next nucleoside triphosphate, by incorporation of nucleoside[1-thio]triphosphate analogues of the four natural RNA nucleosides, and by varying the concentrations of pyrophosphate in the in vitro polymerase reaction. None of these perturbations greatly affected viral RNA polymerase fidelity at the site studied. These results fail to show evidence for proofreading exonuclease activity associated with the virion replicase of an RNA virus. They suggest that RNA virus replication might generally be error-prone, because RNA replicase base misincorporations are proofread very inefficiently or not at all.     

Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway

Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.     

An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction

The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Metal determines efficiency and substrate specificity of the nuclear NUDIX decapping proteins X29 and H29K (Nudt16)

The Xenopus X29 protein was identified by its high affinity binding to U8 small nucleolar RNA, a small nucleolar RNA required for ribosome biogenesis. X29 and its human homologue H29K (Nudt16) are nuclear nucleoside diphosphatase proteins localized within foci in the nucleolus and nucleoplasm. These proteins can remove m(7)G and m(227)G caps from RNAs, rendering them substrates for 5'-3' exonucleases for degradation in vivo. Here, a more complete characterization of these metal-dependent decapping proteins demonstrates that the metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg(+2) the proteins hydrolyze the 5' cap from only one RNA substrate: U8 small nucleolar RNA. However, in the presence of Mn(+2) or Co(+2) all RNAs are substrates and the decapping efficiency is higher. The x-ray crystal structure of X29 facilitated structure-based mutagenesis. Mutation of single amino acids coordinating metal in the active site yielded mutant proteins confirming essential residues. In vitro assays with purified components are consistent with a lack of protein turnover, apparently due to an inability of the protein to release the decapped RNA, implicating critical in vivo interacting factors. Collectively, these studies indicate that the metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs. With the potential broader RNA substrate specificity, X29/H29K may be the nuclear counterparts of the cytoplasmic decapping machinery, localized in specialized bodies involved in RNA decay.