Making ends meet: a model for RNA splicing in fungal mitochondria

Davies, R. Wayne; Waring, Richard B.; Ray, John A.; Brown, Terence A.; Scazzocchio, Claudio

doi:10.1038/300719a0

Cited by 441 publications

(245 citation statements)

References 15 publications

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Section: Introductionmentioning

confidence: 99%

“…Only then PI0 should be allowed to foml. We shall verify this scenario by simulating the folding of the fourth intron of the yeast apocytochrome b gene (YCOB4) [2,3], an intron that requires trunsacting factors to fold into an active structure and overcome its structural deficiencies.The chronological order of events demands a dynamical model of folding where interaction I' engages the internal guiding sequence (IGS) until 5' cleavage disrupts it, precluding the premature formation of PI0 (see Fig. 1).…”

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confidence: 99%

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On how hydrolysis at the 3′ end is prevented in the splicing of a sequentially folded group I intron

Fernández

1992

FEBS Letters

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On how hydrolysis at the 3' end is prevented in the splicing of a sequentially folded group I intron WC propose a dynamic model for the compelition bctwcen exon-exon ligation and 3'-end hydrolysis valid for sequentially folded pre-mRNA introns of group I. This model accounts for the delay in the formation of conserved helix PlO until the 5' exon has been cleaved. a requirement to prevent hydrolysis at the 3' end of the intron. The model is rooted on computer simulations whereby the pre-mRNA searches for its structure as it is being transcribed. Thus, a competing interaction, engging the internal guiding sequence. occurs initially and prevents PlO from forming until the 3'end of the 5' exon is habilitated as a nuclcophilic agent. It is further shown that a destabilization of the compeling interaction invariably leads to 3 hydrolysis, crippling the splicing capability of the intron. The results may be probed by sitedirected mutagen&s.Ribozyme; 3' l-iydrolysis; In vivo RNA folding I. INTRODUCTIONPerhaps the most important reaction competing with exon ligation in the splicing of group I introns is hydrolysis at the 3' end of the intron. The prevention of this competing reaction poses a major difficulty to experimentalists attempting to engineer splicing conditions. Hydrolysis might be precluded if the formation of the conserved helix PlO is delayed until 5' cleavage had taken place and the nucleophilic 3' end of the already spliced 5' exon is ready to attack the 3' intronexon junction [l]. Only then PI0 should be allowed to foml. We shall verify this scenario by simulating the folding of the fourth intron of the yeast apocytochrome b gene (YCOB4) [2,3], an intron that requires trunsacting factors to fold into an active structure and overcome its structural deficiencies.The chronological order of events demands a dynamical model of folding where interaction I' engages the internal guiding sequence (IGS) until 5' cleavage disrupts it, precluding the premature formation of PI0 (see Fig. 1). Thus, the postponement in the formation of PI0 finds concrete meaning in a sequential model for the folding of the pre-mRNA under scrutiny. We advocate that the structure is searched concurrently with the assembling of the intron. In other words, folding is concomitant with transcription and results from the Published by Ehv/er Scicncr Publishers B. V.

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Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

On how hydrolysis at the 3′ end is prevented in the splicing of a sequentially folded group I intron

Fernández

1992

FEBS Letters

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“…Furthermore, the 5′-extension of the IGS forms another helix in the second step of self-splicing, referred to as P10. This helix is formed between the IGS extension and the 3′-exon ( Figure 1A, (40)(41)(42)(43), see also: (44)). Thus, base pairs in the P1 extension must be broken for self-splicing to proceed, and the P1 extension and P10 are dynamic secondary structure elements in self-splicing ( Figure 1A).…”

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confidence: 99%

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

2007

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Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogs, catalyzes a reaction analogous to the first step of self-splicing, in which a 5′-splice site analog (S) and guanosine (G) are converted into a 5′-exon analog (P) and GA. This ribozyme preserves the active site but lacks a short 5′-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5′-splice site in the first step of self-splicing and P10 forms at the 3′-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5′-and 3′-splice site analogs that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogs that do not form additional base pairs with the IGS extension. Analysis of a U•U pair immediately 3′ to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5′-splice site analogs that form only a subset of base pairs reveal that inclusion of the conserved G•U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G•U step. These results suggest a previously unrecognized role of the G•U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.The group I intron from Tetrahymena thermophila, the first catalytic RNA to be discovered, folds into a specific structure and performs a self-splicing reaction that includes chemical steps as well as RNA conformational rearrangements. This reaction is simple, relative to pre-mRNA † This work was supported by NIH grant GM49243. K.K. was supported in part by a predoctoral fellowship from the Boehringer Ingelheim NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript splicing and other RNA-mediated processes, and may serve as a tractable system to learn more about RNA catalysis and its functional conformational change...

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“…By 1985 the sequential steps in RNA selfsplicing had been described (Zaug and Cech 1982) and the secondary structure common to group I introns had been determined (Davies et al 1982;Michel et al 1982;Waring and Davies 1984). Furthermore, several fungal mitochondrial introns and one from bacteriophage T4 had joined the Tetrahymena rRNA intron on the list of self-splicers (Garriga and Lambowitz 1984;Chu et al 1985;van der Horst and Tabak 1985).…”

Section: Introduction Rmentioning

confidence: 99%

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Cech

Herschlag

1996

Nucleic Acids and Molecular Biology

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Making ends meet: a model for RNA splicing in fungal mitochondria

Cited by 441 publications

References 15 publications

On how hydrolysis at the 3′ end is prevented in the splicing of a sequentially folded group I intron

On how hydrolysis at the 3′ end is prevented in the splicing of a sequentially folded group I intron

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

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