Ribonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 5′-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3′ cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G[bull ]U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5′ splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5′ splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5′ end, suggesting that other factors than the U1 snRNA are involved in 5′ splice site recognition. The importance of a consecutive complementarity between the 5′ splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme–substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme–substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-RP phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-SP phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.

A U5 snRNP protein, hPrp8, forms a UV-induced crosslink with the 5′ splice site (5′SS) RNA within splicing complex B assembled in trans- as well as in cis-splicing reactions. Both yeast and human Prp8 interact with the 5′SS, branch site, polypyrimidine tract, and 3′SS during splicing. To begin to define functional domains in Prp8 we have mapped the site of the 5′SS crosslink within the hPrp8 protein. Immunoprecipitation analysis limited the site of crosslink to the C-terminal 50–60-kDa segment of hPrp8. In addition, size comparison of the crosslink-containing peptides generated with different proteolytic reagents with the pattern of fragments predicted from the hPrp8 sequence allowed for mapping of the crosslink to a stretch of five amino acids in the C-terminal portion of hPrp8 (positions 1894–1898). The site of the 5′SS:hPrp8 crosslink falls within a segment spanning the previously defined polypyrimidine tract recognition domain in yPrp8, suggesting that an overlapping region of Prp8 may be involved both in the 5′SS and polypyrimidine tract recognition events. In the context of other known interactions of Prp8, these results suggest that this protein may participate in formation of the catalytic center of the spliceosome.

A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (−)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (−)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (−)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates—similar to in vivo rates—or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.

In vitro transcription with T7 RNA polymerase is one of the most powerful tools in RNA research. This is mainly linked to the easy preparation of the enzyme, the quite unlimited range of sizes and sequences of the RNA that can be synthesized, as well as the efficiency and accuracy of synthesis. So far only two major limitations are common knowledge, namely poor transcription efficiency of non-G-rich initial sequences (Dunn & Studier, 1983) and 3′-end heterogeneities of the transcripts by 1 or 2 nt (Milligan et al., 1987; Draper et al., 1988; Kholod et al., 1998). These drawbacks have been overcome in most cases by improving the initiating sequence and/or purifying the transcription products to single-nucleotide resolution when necessary. The Uhlenbeck laboratory has published the first evidence for shifty errors by the T7 RNA polymerase at the very 5′ end of the synthesized RNA, an as yet unsuspected event (Pleiss et al., 1998). They demonstrated that in the particular case of tRNA sequences starting with G-rich stretches, the enzyme incorporates additional nontemplated G residues in up to 30% of the transcripts. The authors point to the fact that these errors, in combination with the 3′-end heterogeneity common to T7 transcripts, may be crucial, for instance, for functional studies of tRNAs, because some in vitro-transcribed tRNA molecules of rigorously correct final size may be erroneously considered as functional.

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.

Oxazolidinones are antibacterial agents that act primarily against gram-positive bacteria by inhibiting protein synthesis. The binding of oxazolidinones to 70S ribosomes from Escherichia coli was studied by both UV-induced cross-linking using an azido derivative of oxazolidinone and chemical footprinting using dimethyl sulphate. Oxazolidinone binding sites were found on both 30S and 50S subunits, rRNA being the only target. On 16S rRNA, an oxazolidinone footprint was found at A864 in the central domain. 23S rRNA residues involved in oxazolidinone binding were U2113, A2114, U2118, A2119, and C2153, all in domain V. This region is close to the binding site of protein L1 and of the 3′ end of tRNA in the E site. The mechanism of action of oxazolidinones in vitro was examined in a purified translation system from E. coli using natural mRNA. The rate of elongation reaction of translation was decreased, most probably because of an inhibition of tRNA translocation, and the length of nascent peptide chains was strongly reduced. Both binding sites and mode of action of oxazolidinones are unique among the antibiotics known to act on the ribosome.

The assembly of the U4 and U6 snRNPs into the U4/U6 di-snRNP is necessary for pre-mRNA splicing, and in Saccharomyces cerevisiae requires the splicing factor Prp24. We have identified a family of Prp24 homologs that includes the human protein SART3/p110nrb, which had been identified previously as a surface antigen in several cancers. Sequence conservation among the Prp24 homologs reveals the existence of a fourth previously unidentified RNA recognition motif (RRM) in Prp24, which we demonstrate is necessary for growth of budding yeast at 37 °C. The family is also characterized by a highly conserved 12-amino-acid motif at the extreme C terminus. Deletion of this motif in Prp24 causes a cold-sensitive growth phenotype and a decrease in base-paired U4/U6 levels in vivo. The mutant protein also has a reduced association with U6 snRNA in extract, and is unable to interact with the U6 Lsm proteins by two-hybrid assay. In vitro annealing assays demonstrate that deletion of the motif causes a defect in U4/U6 formation by reducing binding of Prp24 to its substrate. We conclude that the conserved C-terminal motif of Prp24 interacts with the Lsm proteins to promote U4/U6 formation.

Analysis of atomic resolution structures of the rRNAs within the context of the 50S and the 30S ribosomal subunits have revealed the presence of nine examples of a recurrent structural motif, first observed in the TΨC loop of tRNAs. The key component of this T-loop motif is a UA trans Watson–Crick/Hoogsteen base pair stacked on a Watson–Crick pair on one side. This motif is stabilized by several noncanonical hydrogen bonds, facilitating RNA–RNA as well as RNA–protein interactions. In particular, the sugar edge of the purine on the 3′ side of the pivotal uridine in the UA pair frequently forms a noncanonical base pair with a distant residue. The bulged-out bases, usually seen as part of the motif, also use their Watson–Crick edges to interact with nearby residues via base-specific hydrogen bonds. In certain occurrences, a backbone reversal is stabilized by specific hydrogen bonds as is observed in the U-turn motifs and the adenosine residue of the key UA pair interacts with a third base via its Watson–Crick edge, essentially generating a base triple.

Although the decoding rules have been largely elucidated, the physical-chemical reasons for the “correctness” of codon:anticodon duplexes have never been clear. In this work, on the basis of the available data, we propose that the correct codon:anticodon duplexes are those whose formation and interaction with the ribosomal decoding center are not accompanied by uncompensated losses of hydrogen and ionic bonds. Other factors such as proofreading, base–base stacking and aminoacyl–tRNA concentration contribute to the efficiency and accuracy of aminoacyl–tRNA selection, and certainly these factors are important; but we suggest that analyses of hydrogen and ionic bonding alone provides a robust first-order approximation of decoding accuracy. Thus our model can simplify predictions about decoding accuracy and error. The model can be refined with data, but is already powerful enough to explain all of the available data on decoding accuracy. Here we predict which duplexes should be considered correct, which duplexes are responsible for virtually all misreading, and we suggest an evolutionary scheme that gave rise to the mixed boxes of the genetic code.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5′ ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5′ and one 3′ purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a “kissing-loop” complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an under-accumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, A1, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.

Aminoacyl-tRNA synthetases attach amino acids to the 3′ termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13–18, 2002) discussed new research into the structure–function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code.

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using “allosteric selection,” a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3′,5′-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062–1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This “affinity maturation” effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5′ splice site upstream of the env open reading frame. To determine the role of this splice site in the 5′-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5′ splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5′ splice site, SD4, and the free 5′ end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5′ splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions −3 to +8 of the 5′ splice site and all 11 nt constituting the single-stranded 5′ end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.

Anticodon hairpins are structural motifs with contradictory functions. The recognition by aminoacyl synthetases implies extended interactions with the anticodon base triplet and thus, usually, an unfolding of the anticodon loop. The recognition by the ribosome and cognate interaction with a mRNA codon implies, on the other hand, the formation of a mini-helix with a canonical anticodon hairpin structure as observed by crystallography and NMR. To be able to understand the various properties of this motif, a precise description of its structural conservation is required. Here, on the basis of phylogenetic, structural, and molecular dynamics data, we discuss a conserved interaction established between the ribose of the U33 and the base at position 35, either a purine or a pyrimidine. This interaction involves the hydrogen bonding donor or acceptor potential of the hydroxyl group of U33 and has to be integrated in an extended definition of the anticodon hairpin. The extended structural signature provides also an explanation for the role played by pseudouridines at position 35.

In this communication, we describe a simple and reliable method for RNA structure determination in vivo, using the divalent ion, lead(II), as a structural probe. Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent. Because the ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50–100 mM lead(II) acetate for 3–7 min, resulting in partial cleavage of RNA in vivo. Cleavage positions were mapped by reverse transcription analysis of total extracted RNA. Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA. All three RNAs had previously been analyzed in vitro, and secondary structure models were available. The results presented here show that lead(II) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work. These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.

The 5′-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882–892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124–1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5′-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5′ cloverleaf RNA and rendered poliovirus mRNA unstable. A 5′-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5′-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5′ cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5′ cloverleaf RNA.