RNase MRP and RNase P are both ribonucleoprotein enzymes performing endonucleolytic cleavage of RNA. RNase MRP cleaves at a specific site in the precursor-rRNA transcript to initiate processing of the 5.8S rRNA. RNase P cleaves precursor tRNAs to create the 5′ end of the mature tRNAs. In spite of their different specificities, the two RNases have significant structural similarities. For example, the two enzymes in Saccharomyces cerevisiae share eight protein subunits; only one protein is unique to each enzyme. The RNA components of the two nucleases also show striking secondary-structure similarity. To begin to characterize the role of the RNA subunits in enzyme function and substrate specificity, we swapped two hairpin structures (MRP3 and P3) between RNase MRP RNA and RNase P RNA of S. cerevisiae. The hairpins in the two enzymes could be exchanged without loss of function or specificity. On the other hand, when the MRP3 hairpin in RNase MRP of S. cerevisiae was replaced with the corresponding hairpin from the RNA of Schizosaccharomyces pombe or human RNase MRP, no functional enzyme was assembled. We propose that the MRP3 and P3 hairpins in S. cerevisiae perform similar functions and have coevolved to maintain common features that are different from those of MRP3 and P3 hairpins in other species.

Members of the serine/arginine-rich (SR) protein family have multiple functions in the pre-mRNA splicing reaction. In addition to being required for the removal of constitutively spliced introns, SR proteins can function to regulate alternative splicing both in vitro and in vivo (Ge & Manley, 1990; Krainer et al., 1990a; Fu et al., 1992; Zahler et al., 1993a; Caceres et al., 1994; Wang & Manley, 1995). In the cell, SR proteins migrate from speckles—subnuclear domains that may function as storage sites for certain splicing factors—to sites of active transcription (Misteli et al., 1997; Misteli & Spector, 1999) and some SR proteins have been found to shuttle in and out of the nucleus (Caceres et al., 1998). The subcellular localization of SR proteins can be modulated by phosphorylation (Misteli & Spector, 1998; Misteli et al., 1998) and this undoubtedly underlies some regulated splicing events. However, once in the nucleus and localized to the nascent pre-mRNA, exactly how SR proteins engage the general splicing machinery to recognize specific splice sites is unclear and is an area of intense investigation.

The problem of mRNA decay in E. coli has recently seen exciting progress, with the discoveries that key degradation enzymes are associated together in a high molecular weight degradosome and that polyadenylation promotes decay. Recent advances make it clear that mRNA decay in bacteria is far more interesting enzymatically than might have been predicted. In-depth study of specific mRNAs has revealed multiple pathways for degradation. Which pathway a given mRNA follows appears to depend in large part on the location of the initiating endonucleolytic cleavage within the mRNA. During the steps of mRNA decay, stable RNA structures pose formidable barriers to the 3′ → 5′ exonucleases. However, polyadenylation is now emerging as a process that plays an important role in maintaining the momentum of exonucleolytic degradation by adding single-stranded extensions to the 3′ ends of mRNAs and their decay intermediates, thereby facilitating further exonuclease digestion.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA–CopT pairing in vitro showed that the dominant product of antisense RNA–mRNA binding is not a full RNA duplex. We have studied here the structure of CopA–CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop–loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA–CopT RNA complex. This stabilizer helix cannot form in CopI–CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI–CopT) and the stable complex (CopA–CopT) are discussed.

Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides. Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions. Here we discuss conserved RNA folding pathways that are revealed by covariation patterns. In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners. Such “coupled” covariations between three or more nucleotides were found in various types of RNAs. The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases. Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts.

The myxomycete Physarum polycephalum requires extensive RNA editing to create functional mitochondrial transcripts. The cytochrome c oxidase subunit 1 (coI) transcript exhibits a combination of editing forms not found together in any other eukaryotic RNA: 66 insertions of ribonucleotides (59 Cs, a single U, and three mixed dinucleotides) as well as base conversion of four Cs to Us (Gott et al., J Biol Chem, 1993, 268:25483–25486). Through a phylogenetic survey of coI DNA genes and RNA transcripts in representative myxomycetes, we have decoupled the four types of editing in this lineage. Some myxomycetes share insertional editing with P. polycephalum, yet lack C → U conversion, consistent with previous reports of separation of insertional and base conversion editing in P. polycephalum extracts (Visomirski-Robic & Gott, RNA, 1995, 3:821–837). Most remarkably, we detect unique evolutionary histories of the three different types of insertional editing, though these have been indistinguishable in vitro. For example, Clastoderma debaryanum exhibits insertions of Us, but not Cs or dinucleotides.

Large ribozymes typically require very long times to refold into their active conformation in vitro, because the RNA is easily trapped in metastable misfolded structures. Theoretical models show that the probability of misfolding is reduced when local and long-range interactions in the RNA are balanced. Using the folding kinetics of the Tetrahymena ribozyme as an example, we propose that folding rates are maximized when the free energies of forming independent domains are similar to each other. A prediction is that the folding pathway of the ribozyme can be reversed by inverting the relative stability of the tertiary domains. This result suggests strategies for optimizing ribozyme sequences for therapeutics and structural studies.

This issue contains three varying perspectives on a specific hypothesis for the origin of the genetic code. RNA is pleased to provide a forum for constructive “debates” such as this one. The editors welcome suggestions for additional topics that could benefit from an open discussion of divergent views.

Translational fidelity, programmed frameshifting, and nonsense-mediated decay (NMD) may require common proteins, reflecting related events in translation and ribosome function (reviewed in Czaplinski et al., 1999). This letter focuses on the possible link between programmed frameshifting and NMD, and seeks to reconcile apparently conflicting conclusions concerning a proposed overlap in the factors involved.

In mammals, as in all organisms examined, mRNAs that prematurely terminate translation are abnormally reduced in abundance by a mechanism called nonsense-mediated mRNA decay (NMD) or mRNA surveillance (for reviews, see Maquat, 1995, 1996; Ruiz-Echevarria & Peltz, 1996; Li & Wilkinson, 1998; Culbertson, 1999; Hentze & Kulozik, 1999; Hilleren & Parker, 1999). This mechanism is thought to have evolved to eliminate nonsense-containing RNAs that arise as a consequence of (1) mutations in germ-line or somatic DNA or (2) routine abnormalities in gene expression due to abnormalities in, for example, transcription initiation, splicing, and somatic rearrangements of the type that characterize the immunoglobulin and T-cell receptor genes. The elimination of nonsense-containing mRNAs protects cells from the potentially deleterious effects of the encoded truncated proteins, which can manifest new or dominant-negative functions (Kazazian et al., 1992; Pulak & Anderson, 1993; Hall & Thein, 1994; Cali & Anderson, 1998). In addition to eliminating abnormal transcripts, NMD also regulates the expression of certain mRNAs that are not abnormal. Examples in mammalian cells are provided by certain selenoprotein mRNAs that terminate translation at a UGA codon for the inefficiently incorporated amino acid selenocysteine (Sec; Moriarty et al., 1997, 1998). Other examples will undoubtedly resemble natural substrates found in other organisms such as the alternatively spliced mRNAs of Caenorhabditis elegans that retain an internal exon harboring an in-frame termination codon (Morrison et al., 1997), and transcripts of Saccharomyces cerevisiae that contain a small open reading frame upstream of the primary open reading frame (Leeds et al., 1992; Pierrat et al., 1993).

It has been known for almost a decade and a half that in trypanosomes all mRNAs are trans-spliced by addition to the 5′ end of the spliced leader (SL) sequence. During the same time period the conviction developed that classical cis-splicing introns are not present in the trypanosome genome and that the trypanosome gene arrangement is highly compact with small intergenic regions separating one gene from the next. We have now discovered that these tenets are no longer true. Poly(A) polymerase (PAP) genes in Trypanosoma brucei and Trypanosoma cruzi are split by intervening sequences of 653 and 302 nt, respectively. The intervening sequences occur at identical positions in both organisms and obey the GT/AG rule of cis-splicing introns. PAP mRNAs are trans-spliced at the very 5′ end as well as internally at the 3′ splice site of the intervening sequence. Interestingly, 11 nucleotide positions past the actual 5′ splice site are conserved between the T. brucei and T. cruzi introns. Point mutations in these conserved positions, as well as in the AG dinucleotide of the 3′ splice site, abolish intron removal in vivo. Our results, together with the recent discovery of cis-splicing introns in Euglena gracilis, suggest that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell.

RNA helices that recapitulate sequences of the tRNA acceptor stem, including the 3′ NCCA nucleotides, can be substrates for aminoacyl–tRNA synthetases (Frugier et al., 1994; Hamann & Hou, 1995; Martinis & Schimmel, 1995; Quinn et al., 1995). Although the catalytic efficiency of aminoacylation of RNA helices is reduced from that of the full-length parent tRNA, the specificity is maintained. The specific aminoacylation lies in the ability of aminoacyl–tRNA synthetases to recognize functional groups within the RNA helices. Analysis of tRNA–synthetase structures has suggested a general principle (Rould et al., 1989; Ruff et al., 1991; Arnez & Moras, 1997). The class I synthetases, which attach an amino acid initially to the 2′-OH of the terminal ribose, approach the acceptor and NCCA end from the minor groove side. The class II synthetases, which attach an amino acid to the terminal 3′-OH, approach from the major groove side (Arnez & Moras, 1997). The class-specific approach leads to tRNA–synthetase complexes that are near mirror images of each other and provides a structural rationale for the stereochemistries of aminoacylation. We report here the identification of a functional group in the acceptor end of Escherichia coli tRNACys that is important for the class I cysteine–tRNA synthetase. This functional group makes one of the largest energetic contributions to aminoacylation. However, it is located on the major groove side of the acceptor stem. Kinetic analysis of the contribution of this functional group to aminoacylation suggests new features that are not anticipated from the class-specific approach of synthetases.

Uridylate residues (Us) are inserted and deleted at precise positions in mitochondrial transcripts of Trypanosoma brucei. These sequence changes are determined by interactions with small guide RNAs (gRNAs) that are complementary to edited sequence. Adenylate (A) and guanylate (G) residues in gRNAs across from editing sites pair with inserted Us. We evaluate whether sequence bias exists in the bases surrounding insertion sites. Upon analyzing all reported insertion sites in T. brucei, we find that the predicted base pairs flanking insertion sites show a strong bias. Specifically, guiding As and Gs tend to be flanked by cytosine residues and Us. This bias is expected if precise base-pair interactions at the editing site determine the number of inserted Us.

The complexity of the nonsense-mediated decay (NMD) system makes it difficult to study by comparing the expression of various single reporter constructs. The known effects of the NMD genes include a reduction both in mRNA stability (reviewed by Czaplinski et al., 1999) and in the efficiency of translational initiation (Muhlrad & Parker, 1999) of nonsense-containing plasmids as well as an apparent increase in the efficiency of translational termination as evidenced by increased readthrough of nonsense mutations (Bidou et al., 2000; Maderazo et al., 2000). The single reporter system can not distinguish among these effects and inference is required to determine which mechanism underlies any observed phenotypic effect on gene expression. It is particularly problematic to differentiate the effects of translation initiation accuracy from putative effects on translational frameshifting. The dual reporter system used in our work isolates the effect of translational frameshifting from effects on mRNA stability, initiation or termination. Much is made by Dinman et al. of the relative effects of various mutations, yet it remains unclear whether these are fundamental differences or simply differences in phenotypic strength of the various mutations.

An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2′ deoxyribose substitutions in binding-site residues were tested for theophylline-binding affinity and the results were compared with the previously determined three-dimensional structure of the RNA–theophylline complex. The RNA–theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2′-deoxy A28 mutant reduces theophylline binding >45-fold at 25 °C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2′-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the “conserved core” of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg2+, Mn2+, or Co2+ ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn2+-induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer.

The wobble GoU pairs have been implicated in several biological processes where RNA molecules play a key role. We review the geometrical and conformational properties of wobble GoU pairs on the basis of available crystal structures of RNAs at high resolution. The similarities with the wobble A+oC pairs and UoU pairs are illustrated, while the differences with the recently discovered bifurcated G[bull ]U pairs are contrasted.

The inactivity or occlusion of the HIV-1 poly(A) signal when in the 5′ long terminal repeat (LTR) has been mechanistically investigated. First we show that neither the homologous HIV-1 promoter nor the close proximity of this RNA processing signal to the transcript initiation site is required for the occlusion effect. Instead we demonstrate that the major splice donor (MSD) site positioned about 200 bp downstream maintains the poly(A) site in an inactive state. Although mutation of MSD results in activation of the 5′ LTR poly(A) signal, this effect can be suppressed by targeting U1 snRNAs near to the mutated MSD by base pairing. We show that hybrid U7-U1 snRNAs can also suppress the poly(A) signal and that this suppression is dependent on the U1 stem-loop 1. In particular the binding site for the U1 snRNP protein 70K that binds to the loop structure of stem-loop 1 is associated with poly(A) site occlusion. These experiments were carried out with an HIV-1 proviral construct and as such emphasize the physiological importance of this splice donor–poly(A) site interaction.

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)63+) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)63+ is a useful spectroscopic probe for Mg(H2O)62+-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)63+. We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C[bull ]G base pair is replaced by U[bull ]G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3′ direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)63+ and RNA protons were used to locate the site of Co(NH3)63+ binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5′ by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.

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.

The crystal structure of the monoclinic form of yeast phenylalanine tRNA has been redetermined at a resolution of 1.93 Å. The structure of yeast tRNAphe described here is more accurate than its predecessors not only because it incorporates higher resolution data, but also because it has been refined using techniques that had not been developed when its predecessors were determined more than 20 years ago. The 1.93 Å resolution version of this structure differs interestingly from its predecessors in its details. In loop regions particularly, the backbone torsion angles in the new structure are not the same as those reported earlier. Several new divalent cation binding sites have been identified, and the water structure that has emerged is also different.