I. INTRODUCTION During morphogenesis, some cells migrate from their origins to distant locations. The complex, stereotyped migrations of axonal growth cones, for example, determine the connectivity of the nervous system. Motile cells and processes can undergo directed movements in response to spatially patterned molecules (Keynes and Cook 1995; Garrity and Zipursky 1995), and recent studies suggest that specific directional cues are conserved from nematodes to chordates (Hedgecock et al. 1990; Ishii et al. 1992; Kennedy et al. 1994; Serafini et al. 1994; Colamarino and Tessier-Lavigne 1995). Migrating cells may follow several different directional cues, often in strict sequence, during development. Moreover, they may halt migration at stereotyped positions or times either to divide or to differentiate. These changes in cell movements must reflect changes in the extracellular environment or changes within the motile cell itself. For example, cells might be constitutively responsive to multiple directional cues, changing course whenever they encounter a further cue within their repertoire. Instead, it appears that cells become responsive to new cues, or unresponsive to current ones, during the course of migration. Some transitions could be entirely autonomous, reflecting, for example, an intracellular clock, but many are contingent upon extracellular signals encountered during the course of migration. These transitions often involve new gene expression. Analysis of these transitions has identified signaling pathways and transcriptional cascades within migrating cells that help select substrate and direction of migration, or regulate transitions between motile and stationary states. One such pathway in nematodes responds to hormonal signals that advance...

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.

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.

We have identified nuclear mutants of Neurospora that are defective in splicing the mitochondrial large rRNA and that accumulate unspliced pre-rRNA (35S RNA). In cyt-4 mutants, the unspliced pre-rRNA contains short 3′ end extensions (110 nucleotides) that are not present in pre-rRNAs from the other mutants. This and other characteristics suggest that the cyt-4 mutants may be primarily defective in 3′ end synthesis and the RNA splicing defect occurs secondarily as a result of impaired RNA folding. The cyt-4 mutants also accumulate a “short” intron RNA and small axon RNAs that may reflect aberrant RNA cleavages. The 5′ end of the short intron is about 285 nucleotides downstream from the 5′ splice site at or near the base of the “central hairpin”, a putative intermediate in folding of the pre-rRNA. Furthermore, the aberrant cleavage sites are immediately after a six nucleotide sequence (GAUAAU) homologous to the final splice junction (GAU/AAC).

We have used Neurospora nuclear mutant cyt-18-1, which accumulates a number of unspliced mitochondrial precursor RNAs, to identify rapidly mitochondrial introns that are self-splicing in vitro. Incubation of deproteinized whole mitochondrial RNA from the mutant with 32P-GTP resulted in strong labeling of a 1.3 kb RNA, subsequently identified as cytochrome b (cob) intron 1, and weaker labeling of additional RNAs. Self-splicing of cob intron 1, including precise cleavage and ligation, was confirmed using an in vitro transcript synthesized from the SP6 promoter. The in vitro splicing reaction was shown to be analogous to that for the Tetrahymena nuclear rRNA intron. Since splicing of cob intron 1 is inhibited in a recessive nuclear mutant, we infer that this essentially RNA-catalyzed splicing reaction must be facilitated by a protein in vivo.

The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5' end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.

In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal RNA contains an intervening sequence of 2.3 kb. We have identified eight nuclear mutants that are defective in splicing the mitochondrial large ribosomal RNA and that accumulate unspliced precursor RNA. These mutants identify three different nuclear genes required for the same mitochondrial RNA splicing reaction. Some of the mutants have unique phenotypic characteristics (for example, accumulation of an unusual intron RNA) that may provide insight into specific aspects of mitochondrial RNA splicing. Mutations at one locus, cyt4, are subject to partial phenotypic suppression by the electron-transport inhibitor antimycin. This phenomenon suggests that at least one component required for mitochondrial RNA splicing is regulated such that its synthesis or activity is increased in response to impairment of electron transport.

The mtDNAs of lower and higher eukaryotes contain essentially the same complement of genes coding rRNA and tRNA species and a limited number of inner membrane polypeptides, but they show considerable divergence in size, structure, and genetic organization (Gillham 1978). In fungi the genes coding the small and large mitochondrial rRNAs are separated by stretches of DNA ranging from 1.3 kbp to 30 kbp, whereas in animal mtDNAs the corresponding genes are separated by less than 200 bp (Boynton et al. 1980; Grant and Lambowitz 1982). In addition, some yeast and fungal mtDNAs, but not animal mtDNAs, contain intervening sequences in the genes that code certain polypeptides (Borst and Grivell 1978) and the large rRNA (Boynton et al. 1980; Grant and Lambowitz 1982). Although the basic features of mtDNA gene organization have now been elucidated for some animals and lower eukaryotes, there is still relatively little information about the regulation of mitochondrial gene expression in any experimental system. Such questions are best addressed by biochemical-genetic approaches in lower eukaryotes. We have been developing an experimental system focusing on nuclear mutants of Neurospora crassa that are defective in splicing the mitochondrial large rRNA. The mutants open unique experimental approaches for studying RNA splicing and its role in gene expression. In this paper we review the salient features of the experimental system and the mutants. In addition, we present evidence, from the characterization of one mutant, that RNA splicing in Neurospora mitochondria may be subject to regulation by the rate of electron...