The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns in mitochondria. Here, we show that CYT-18 binds strongly to diverse group I introns that have minimal sequence homology and recognizes highly conserved structural features of the catalytic core of these introns. Inhibition experiments indicate that the intron RNA and tRNA TM compete for the same or overlapping binding sites in the CYT-18 protein. Considered together with functional analysis, our results indicate that the CYT-18 protein promotes splicing by binding to the intron core and stabilizing it in a conformation required for catalytic activity. Furthermore, the specific binding of the synthetase suggests that the group I intron catalytic core has structural similarities to tRNAs, which could reflect either convergent evolution or an evolutionary relationship between group I introns and tRNAs.[Key Words: Catalytic RNA; group I intron; RNA splicing; aminoacyl-tRNA synthetase; mitochondrial RNA processing]Received April 14, 1992; revised version accepted June 1, 1992.Although group I introns use RNA-catalyzed splicing reactions, many, if not all, require proteins for efficient splicing in vivo (for review, see Lambowitz and Perlman 1990). In the case of mitochondrial RNA splicing, these proteins have been shown to include maturases encoded within some group I introns, as well as a variety of splicing factors encoded by host chromosomal genes. The latter tend to differ between Neurospora and yeast, and they include aminoacyl-tRNA synthetases and other cellular RNA-binding proteins that have an additional function in their host cell. We suggested that host proteins that have already differentiated may have adapted to function in splicing relatively recently in evolution by taking advantage of their ability to recognize structural features of group I introns resembling those in their normal cellular RNA targets (Lambowitz and Perlman 1990). Most host-encoded splicing factors function in splicing one or a small number of related group I introns. However, the Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), functions in splicing a number of different group I introns in mitochondria and presumably recognizes conserved structural features of these introns (Collins and Lambowitz 1985). The fact that the CYT-18 protein is an aminoacyl-tRNA synthetase raises the possibility that 1Corresponding author. these structural features resemble those found in tRNAs (Akins and Lambowitz 1987).To investigate how the Neurospora mt TyrRS functions in splicing, we developed an in vitro-splicing system based on the ability of the protein to splice the Neurospora mitochondrial large rRNA (mt large rRNA) intron, which is not self-splicing under any conditions examined (Garriga and Lambowitz 1986). Studies using this in vitro system showed that splicing occurs by the same guanosine-initiated transesterification reactions used by self-splicing group I introns and remains dependent on the integr...

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