Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme

Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (EDeltaP5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and EDeltaP5abc RNAs is exceptionally strong, with a Kd of approximately 100 pM at 10 mM MgCl2 (at 37 degrees C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.     

The P5abc peripheral element facilitates preorganization of the tetrahymena group I ribozyme for catalysis

Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-function analysis to investigate the role of one of these elements, P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. Structural comparisons using solution-based free radical cleavage revealed that a ribozyme lacking P5abc (E(DeltaP5abc)) and E(DeltaP5abc) with P5abc added in trans (E(DeltaP5abc).P5abc) adopt a similar global tertiary structure at Mg(2+) concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E(DeltaP5abc) is greatly compromised in overall oligonucleotide cleavage activity, even at Mg(2+) concentrations as high as 100 mM. Further characterization of E(DeltaP5abc) via DMS modification revealed local structural differences at several positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified defects in binding of both substrates to E(DeltaP5abc), with > or =25-fold weaker binding of a guanosine nucleophile and > or =350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10(4)-fold decrease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.     

Protein Roles in Group I Intron RNA Folding: The Tyrosyl-tRNA Synthetase CYT-18 Stabilizes the Native State Relative to a Long-Lived Misfolded Structure without Compromising Folding Kinetics

The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally similar misfolded conformation that has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E^ΔP5abc). Here, we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E^ΔP5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, similar to P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by dimethyl sulfate footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structural features, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native contacts as they form. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics.     

Quantifying the energetic interplay of RNA tertiary and secondary structure interactions

To understand the RNA-folding problem, we must know the extent to which RNA structure formation is hierarchical (tertiary folding of preformed secondary structure). Recently, nuclear magnetic resonance (NMR) spectroscopy was used to show that Mg2+-dependent tertiary interactions force secondary structure rearrangement in the 56-nt tP5abc RNA, a truncated subdomain of the Tetrahymena group I intron. Here we combine mutagenesis with folding computations, nondenaturing gel electrophoresis, high-resolution NMR spectroscopy, and chemical-modification experiments to probe further the energetic interplay of tertiary and secondary interactions in tP5abc. Point mutations predicted to destabilize the secondary structure of folded tP5abc greatly disrupt its Mg2+-dependent folding, as monitored by nondenaturing gels. Imino proton assignments and sequential NOE walks of the two-dimensional NMR spectrum of one of the tP5abc mutants confirm the predicted secondary structure, which does not change in the presence of Mg2+. In contrast to these data on tP5abc, the same point mutations in the context of the P4-P6 domain (of which P5abc is a subdomain) shift the Mg2+ dependence of P4-P6 folding only moderately, and dimethyl sulfate (DMS) modification experiments demonstrate that Mg2+ does cause secondary structure rearrangement of the P4-P6 mutants' P5abc subdomains. Our data provide experimental support for two simple conclusions: (1) Even single point mutations at bases involved only in secondary structure can be enough to tip the balance between RNA tertiary and secondary interactions. (2) Domain context must be considered in evaluating the relative importance of tertiary and secondary contributions. This tertiary/secondary interplay is likely relevant to the folding of many large RNA and to bimolecular snRNA-snRNA and snRNA-intron RNA interactions.     

New pathways in folding of the Tetrahymena group I RNA enzyme.

Previous studies have shown that the earliest detectable step in folding of the Tetrahymena ribozyme is tertiary structure formation of the peripheral element P5abc. This, along with other results, has suggested that P5abc may serve as a scaffold upon which additional tertiary structure is built. Herein we use the onset of oligonucleotide cleavage activity as a readout for native state formation and investigate the effect of P5abc on the rate of folding to the native structure. Despite the early folding of P5abc, its removal to give the E delta P5abc variant decreases the rate of attainment of an active structure less than fivefold (20-100 mM Mg2+, 15-50 degrees C). Furthermore, P5abc added in trans is able to bind the folded E delta P5abc ribozyme and promote oligonucleotide cleavage at least tenfold more rapidly than folding of the wild-type ribozyme, indicating that E delta P5abc does not have to first unfold before productively binding P5abc to form the true native state. This suggests that a state with the overall tertiary structure formed but with P5abc unfolded represents a viable on-pathway intermediate for the wild-type ribozyme. These results provide strong evidence for the existence of two pathways to the native state: in one pathway P5abc forms tertiary structure first, and in another it forms late. The pathway in which P5abc forms first is favored because P5abc can fold quickly and because its tertiary structure is stable in the absence of additional structured elements, not because P5abc formation is required for subsequent folding steps. In the course of these experiments, we also found that most of the ribozyme population does not reach the native state directly under standard conditions in vitro, but instead forms an inactive structure that is stable for hours. Finally, the fraction that does fold to the native state folds with a single rate constant of 1 min-1, suggesting that there are no significantly populated "fast-track" pathways that reach the native state directly by avoiding slow folding steps.     

Folding mechanism of the Tetrahymena ribozyme P4-P6 domain

Synchrotron X-ray-dependent hydroxyl radical footprinting was used to probe the folding kinetics of the P4-P6 domain of the Tetrahymena group I ribozyme, which forms a stable, closely packed tertiary structure. The 160-nt domain folds independently at a similar rate (approximately 2 s(-1)) as it does in the ribozyme, when folding is measured in 10 mM sodium cacodylate and 10 mM MgCl(2). Surprisingly, tertiary interactions around a three-helix junction (P5abc) within the P4-P6 domain fold at least 25 times more rapidly (k >/= 50 s(-1)) in isolation, than when part of the wild-type P4-P6 RNA. This difference implies that long-range interactions in the P4-P6 domain can interfere with folding of P5abc. P4-P6 was observed to fold much faster at higher ionic strength than in 10 mM sodium cacodylate. Analytical centrifugation was used to measure the sedimentation and diffusion coefficients of the unfolded RNA. The hydrodynamic radius of the RNA decreased from 58 to 46 A over the range of 0-100 mM NaCl. We propose that at low ionic strength, the addition of Mg(2+) causes the domain to collapse to a compact intermediate where P5abc is trapped in a non-native structure. At high ionic strength, the RNA rapidly collapses to the native structure. Faster folding most likely results from a different average initial conformation of the RNA in higher salt conditions.     

Deletion of the P5abc peripheral element accelerates early and late folding steps of the Tetrahymena group I ribozyme

The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant E(deltaP5abc) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E(deltaP5abc) forms its earliest stable tertiary structure on the millisecond time scale, approximately 5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E(deltaP5abc) in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E(deltaP5abc) ribozyme is much slower (approximately 0.6 min(-1)), in a manner similar to that of the wild type. Also similar, only a small fraction of E(deltaP5abc) attains the native state on this time scale under standard conditions at 25 degrees C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E(deltaP5abc) refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.     

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

RBFOX1 and RBFOX2 are alternative splicing factors that are predominantly expressed in the brain and skeletal muscle. They specifically bind the RNA element UGCAUG, and regulate alternative splicing positively or negatively in a position-dependent manner. The molecular basis for the position dependence of these and other splicing factors on alternative splicing of their targets is not known. We explored the mechanisms of RBFOX splicing activation and repression using an MS2-tethering assay. We found that the Ala/Tyr/Gly-rich C-terminal domain is sufficient for exon activation when tethered to the downstream intron, whereas both the C-terminal domain and the central RRM are required for exon repression when tethered to the upstream intron. Using immunoprecipitation and mass spectrometry, we identified hnRNP H1, RALY, and TFG as proteins that specifically interact with the C-terminal domain of RBFOX1 and RBFOX2. RNA interference experiments showed that hnRNP H1 and TFG modulate the splicing activity of RBFOX1/2, whereas RALY had no effect. However, TFG is localized in the cytoplasm, and likely modulates alternative splicing indirectly.     

The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.     

Trans-activation of the Tetrahymena group I intron ribozyme via a non-native RNA-RNA interaction

The peripheral P2.1 domain of the Tetrahymena group I intron ribozyme has been shown to be non-essential for splicing. We found, however, that separately prepared P2.1 RNA efficiently accelerates the 3' splice-site-specific hydrolysis reaction of a mutant ribozyme lacking both P2.1 and its upstream region in trans. We report here the unusual properties of this trans-activation. Compensatory mutational analysis revealed that non-native long-range base-pairings between the loop region of P2.1 RNA and L5c region of the mutant ribozyme are needed for the activation in spite of the fact that P2.1 forms base-pairings with P9.1 in the Tetrahymena ribozyme. The trans -activation depends on the non-native RNA-RNA interaction together with the higher order structure of P2.1 RNA. This activation is unique among the known trans-activations that utilize native tertiary interactions or RNA chaperons.     

The legumin gene family: structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element.

The field bean, Vicia faba L. var. minor, possesses two sub-families of 11 S legumin genes named A and B. We isolated from a genomic library a B-type gene (LeB4) and determined its primary DNA sequence. Gene LeB4 codes for a 484 amino acid residue prepropolypeptide, encompassing a signal peptide of 22 amino acid residues, an acidic, very hydrophilic alpha-chain of 281 residues and a basic, somewhat hydrophobic beta-chain of 181 residues. The latter two coding regions are immediately contiguous, but each is interrupted by a short intron. Type A legumin genes from soybean and pea are known to have introns in the same two positions, in addition to an extra intron (within the alpha-coding sequence). Sequence comparisons of legumin genes from these three plants revealed a highly conserved sequence element of at least 28 bp, centered at approximately 100 bp upstream of each cap site. The element is absent from the equivalent position of all non-legumin and other plant and fungal genes examined. We tentatively name this element "legumin box" and suggest that it may have a function in the regulation of legumin gene expression.     

Regulation of the human beta-actin promoter by upstream and intron domains.

We have identified three regulatory domains of the complex human beta-actin gene promoter. They span a region of about 3000 bases, from not more than -2011 bases upstream of the mRNA cap site to within the 5' intron (832 bases long). A distal upstream domain contains at least one enhancer-like element. A proximal upstream domain, with a CArG [for CC(A + T rich)6GG] motif found in all known mammalian actin genes, seems to confer serum, but not growth factor, inducibility. The third domain is within the evolutionarily conserved 3' region of the first intron and contains a 13 base-pair sequence, identical to the upstream sequence with the CArG motif. This domain also contains sequences that are both serum and fibroblast growth factor inducible.