RNA splicing in Neurospora mitochondria : Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1

Abstract

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b ( cob) gene, which contains two introns, and a “long form” of the cytochrome oxidase subunit I ( coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of col intron 1, which is excised as a 2.6 × 10 3 base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 °C to 37 °C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18-1 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.