Abstract
The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.
Highlights
The gene encoding the Neurospora mitochondrial spora mt large rRNA intronis spliced by the sameguanosinelarge rRNA contains a single group I intron of 2.3 initiatedtransesterificationreactions used by self-splicing kilobases that is not self-splicing in vitro.We showed group I introns, but in this case, splicing is dependent upon previously that the splicing of this intron in vivo and proteins, which are presumablyrequired for correct folding of in vitro is dependent on the Neurospora cyt-18 pro- the precursor RNA (Garriga anLdambowitz, 1986)
Our findings indicate that the inability of the in the cyt-18 mutants enabled us to analyze its structure by mitochondrial large rRNA intron steolf-splice reflects direct experimental methods andto compare the structureof deficiency of a structure or activity required for cleav-deproteinized 35 S pre-rRNA with that of 35 S pre-rRNA age at the5‘ splice site, either in the intron coriteself assembled in R N P particles (Grimm et al, 1981; Wollenzien or in the interaction between the core and Pt1hestem. et al, 1983)
Our results suggest that the inability of the Neurospora mt large rRNA intron to selfsplice reflects deficiency of a structure or activity required for the 5’ splice site cleavage reaction, either in the intron core itself or in the interaction between the core and theP1 stem
Summary
Secondary Structure Model of the mt Large rRNA IntronFig. 1shows a proposed secondary structure of the Neurospora mt large rRNA intron. The 5' ends of the resulting RNase 111 fragments were mapped by the S1 nuclease procedure, using a 5' end-labeled AccIIHincII fragment (1742 bp) For this initial experiment, we chose a relatively long fragment that encompasses the region expected to contain the central hairpin. The sequence of the intron was determined by Burke and RajBhandary (1982), but has three corrections based on our sequencing: U,,, has been added, C:SW(previously C,,,) has been changed to U, and (previously Azzs0) has been deleted.
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