Ribonucleic acid splicing in Neurospora Mitochondria: secondary structure of the 35S ribosomal precursor ribonucleic acid investigated by digestion with ribonuclease III and by electron microscopy.

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In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal ribonucleic acid (rRNA) contains an intervening sequence of approximately 2.3 kilobases (kb). We have identified two temperature-sensitive mutants (289-67 and 299-9) which are defective in a factor encoded by a nuclear gene but required for the splicing of 25S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants accumulate a novel 35S RNA (5.2-5.6 kb) which is related to the natural precursor of 25S RNA and which has been shown to be a collinear transcript of the 25S RNA gene including the intervening sequence. In the present work, the secondary structure of 35S RNA was investigated by digestion with ribonuclease III and by electron microscopy of the RNA spread under partially denaturing conditions. Ribonuclease III cleaves 35S RNA predominantly at a central site or sites near the 5'-intron-exon boundary and produces fragments which correspond roughly to half-molecules (2.5-3 kb). Electron microscopy of 35S RNA shows a relatively large, central hairpin (180 +/- 45 nucleotides), which presumably corresponds to the central ribonuclease III site, and few other secondary structure features. Both experimental approaches indicate that the large hairpin is not present in 35S RNA. From this finding and from the location of the hairpin near the 5'-intron-exon boundary in 35S RNA, we infer that its formation requires intron sequences. 35S RNA from the mutants can be isolated as a ribonucleoprotein particle associated with almost the full complement of large subunit ribosomal proteins. The 35S RNA in such particles can be cleaved by ribonuclease III at the central site(s), consistent with the idea that the central hairpin is accessible to RNA-processing enzymes in vivo.