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PubMed1996

Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.

Animals↗

PubMed1995

Horizontal transmission of group-I ribozymes: viruses as a carrier of the introns.

The nucleotide sequences of two self-splicing group I introns found in viruses that infect the eukaryotic green alga Chlorella were compared with those of 19 nuclear group I introns of 11 green algae, 1 red alga, 2 yeasts and 1 protist. The similarity ranging from 69.5% to 89.0% in any comparison strongly suggested a close relatedness of these introns. Since this kind of viruses infect algal cells residing in protists such as Paramecium, they may serve as a mediator to spread group I introns horizontally among organisms of different phylogenetic kingdoms.

Animals↗

PubMed1996

X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering.

For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.

Animals↗

PubMed1997

Characterization of Neurospora mitochondrial group I introns reveals different CYT-18 dependent and independent splicing strategies and an alternative 3' splice site for an intron ORF.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing the N. crassa mitochondrial large rRNA intron by stabilizing the catalytically active structure of the intron core. Here, a comprehensive study of N. crassa mtDNA group I introns identified two additional introns, cob-I2 and the ND1 intron, that are dependent on CYT-18 for splicing in vitro and in vivo. The other seven N. crassa mtDNA group I introns are not CYT-18-dependent and include five that self-splice and two that do not splice under any conditions examined. Some of these introns may require maturases or other proteins for efficient splicing. All but one of the non-CYT-18-dependent introns contain large peripheral extensions of the P5 stem, related to the P5abc structure that blocks CYT-18 binding to the Tetrahymena large rRNA intron. The remaining non-CYT-18-dependent intron, cob-I1, contains a long, peripheral extension of the P9 stem, denoted P9.1, which also impedes CYT-18 binding. Detailed analysis of the CYT-18-dependent ND1 intron showed that two 3' splice sites are used in vitro and in vivo. The proximal, alternative 3' splice site brings the intron open reading frame, which potentially encodes a mobility endonuclease, in frame with the upstream exon, possibly providing a means of expression. Considered together, our results show that group I introns in N. crassa mitochondria use a variety of strategies involving different proteins and/or RNA structures to assist splicing, and they support the hypothesis that CYT-18 and the peripheral RNA structure P5abc are alternative evolutionary adaptations for stabilizing the active structure of the intron core.

Alternative Splicing↗

PubMed1997

RNA-RNA interaction and gene splicing.

The precise excision of intervening sequences during RNA splicing is an interesting example of the high degree of specificity involved in biosynthesis processes. Self-splicing RNA precursors achieve this specificity primarily through intramolecular interactions whereas all other types of RNA splicing requires interaction between cellular factors and specific recognition signals in the RNA precursor. About twelve years ago, the in vitro splicing system was developed and a general scheme of the pre-mRNA was proposed (Hernandez and Keller, 1983; Krainer et al., 1984; Lin et al., 1985; Padgett et al., 1984; Ruskin et al., 1984). A fundamental question in the splicing field is how the 5' and 3' splice sites are recognized and paired during the splicing reaction. Recent work in the splicing field has established that a network of RNA interactions may form the structural foundation of the spliceosomes. Possible solutions to many unsolved puzzles are getting attention. RNA-RNA interactions now appear to underlie many aspects of substrate recognition, reaction partner juxtaposition and catalysis. In this article we have presented the latest mechanisms involved in the pre-mRNA splicing and their implication in applied research including cancer.

RNA↗

PubMed1997

An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis.

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule.

Base Sequence↗

PubMed1997

Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron.

Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.

Adenosine↗

PubMed1997

Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes.

Tertiary interactions are important in the higher-order folding of catalytic RNAs. Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanobacterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila. This base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7. We made base pair and single-nucleotide substitutions in the fifth base pair of P4, a G-C in the wild-type Anabaena intron, and tested them for self-splicing activity. The results suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7. Comparative sequence analysis was used to determine the different combinations of base triples that occur in approximately 450 natural group I introns identified to date. About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model. Disrupting this base triple in the Tetrahymena intron resulted in the disappearance of splicing intermediates (intron 3' exon and 5' exon), even though the first step of splicing was not affected. Restoration of the base triple by a compensatory mutation reverted the intermediates to wild-type levels. These results suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding the second step. Repositioning of the base triple to form a new set of interactions may be required for the second step of splicing.

Anabaena↗

PubMed2006

Conformation of the Group II intron branch site in solution.

Group II introns are multidomain ribozymes that catalyze their own removal from pre-mRNA. The nucleophile for the first cleavage step is the 2'OH of a specific adenosine within domain 6 (D6), called the branch site. Mechanistic parallels and limited secondary structural similarity with the eukaryotic spliceosome lead many to speculate that the two systems have a common ancestry. We have elucidated structural features of the branch site region and the importance of the internal loop to branch site conformation within D6 of the ai5gamma Group II intron by NMR and fluorescence spectroscopy. Fluorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to solvent and that this position is enhanced by Mg2+ or Ca2+. Upfield NMR chemical shifts of imino protons of the two uridine residues flanking the branch site adenosine, and an n --> n + 2 NOE between them, suggest a stacked intrahelical conformation of the two uridines. In contrast, results of NMR and 2-aminopurine fluorescence spectra of a mutated D6 from which the internal loop had been deleted suggest a less exposed position of the branch site adenosine, which is likely to form a G-A base pair with the opposing 3'G. These findings describe a model in which the branch site adenosine of D6 is in an extrahelical position, surrounded by two intrahelical bases. The internal loop and divalent metal ions facilitate this motif.

2-Aminopurine↗

PubMed2006

A folding control element for tertiary collapse of a group II intron ribozyme.

Ribozymes derived from the group II intron ai5gamma collapse to a compact intermediate, folding to the native state through a slow, direct pathway that is unperturbed by kinetic traps. Molecular collapse of ribozyme D135 requires high magnesium concentrations and is thought to involve a structural element in domain 1 (D1). We used nucleotide analog interference mapping, in combination with nondenaturing gel electrophoresis, to identify RNA substructures and functional groups that are essential for D135 tertiary collapse. This revealed that the most crucial atoms for compaction are located within a small section of D1 that includes the kappa and zeta elements. This small substructure controls specific collapse of the molecule and, in later steps of the folding pathway, it forms the docking site for catalytic D5. In this way, the stage is set for proper active site formation during the earliest steps of ribozyme folding.

Base Sequence↗

PubMed2005

An obligate intermediate along the slow folding pathway of a group II intron ribozyme.

Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135. We find that structural collapse and native folding of Domain 1 precede assembly of the entire ribozyme, indicating that D1 contains an on-pathway intermediate to folding of the D135 ribozyme. Subsequent docking of Domains 3 and 5, for which D1 provides a preorganized scaffold, appears to be very fast and independent of one another. In contrast to other RNAs, the D135 ribozyme undergoes slow tertiary collapse to a compacted state, with a rate constant that is also limited by the formation D1. These findings provide a new paradigm for RNA folding and they underscore the diversity of RNA biophysical behaviors.

Base Sequence↗

PubMed2006

Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA.

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g. three of the five group I introns in Chlamydomonas reinhardtii. We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii (Cr.LSU), by determining if the inhibition by Mn2+ involves catalysis, folding, or both. Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5 Delta Gb, that lacks the 3' exon and intron-terminal G shows that Mn2+ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme. Surprisingly, ribozyme misfolding induced by Mn2+ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg2+. At lower Mn2+ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP). The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn2+. Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5 Delta Gb was examined using Fe2+-EDTA cleavage. The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3-P7 domain and the nucleotides that connect it to the P4-P5-P6 domain are exposed to solvent. The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed.

Animals↗