Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.

Abstract

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987 Group I introns include nuclear rRNA introns of Tetrahymena spp. and Physarum polycephalum, most fungal mitochondrial DNA introns, some chloroplast introns, and introns in T-even bacteriophages (5). All group I introns appear to use the same splicing mechanism first elucidated for the Tetrahymena thermophila nuclear rRNA intron by T. R. Cech and co-workers. This mechanism involves two sequential transesterification reactions: addition of guanosine to the 5' end of the intron coupled to cleavage at the 5' splice site and exon ligation coupled to cleavage at the 3' splice site (5). Some group I introns, including the Tetrahymena nuclear rRNA intron, some mitochondrial DNA introns in Neurospora crassa and Saccharomyces cerevisiae, and introns in T-even bacteriophage, are efficiently self splicing in vitro (5). Self-splicing indicates that both the structural information and catalytic activities required for splicing are contained in the structure of the intron RNAs. The basic outline of the catalytically active structure of group I introns has been elucidated by phylogenetic comparisons, analysis of in vivo and in vitro mutants, and direct RNA structure analysis (5). All a core secondary structure that includes base pairing between short sequence elements P3[5'], P, Q, R, P3[3'], and S. Group I introns also contain an internal guide sequence that base pairs with flanking sequences in the 5' exon to position the 5' splice site for splicing and may also play a role in positioning the 3' splice site (4, 5). The internal region of the intron, including the core structure, possesses the catalytic activities that cleave at the 5' splice site and add guanosine to the 5' end of the intron (35). In those group I introns that are self-splicing in vitro, the catalytically active structure must be favored relative to alternative structures of deproteinized precursor RNAs. Although some group I introns are self-splicing in vitro, genetic analysis has shown that most, if not all, group I introns are dependent upon proteins for splicing in vivo. The most likely hypothesis is that these proteins function to fold the RNA into the catalytically active conformation. We showed previously that N. crassa cob intron 1, which is efficiently self-splicing in vitro, is dependent on the protein encoded by the cyt-18 gene for splicing in vivo (6,12). The N. crassa mitochondrial large rRNA intron has been shown to require proteins for splicing both in vivo and in vitro (13).The proteins required for splicing group I introns can be divided into three classes: (i) maturases, a family of structurally related proteins encoded within some, but not all, group I introns (26, 36); (ii) intron-specific proteins encoded by nuclear genes, e.g., the CBP2 and MRS1 proteins of S. cerevisiae, whic...

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

“…Interestingly, the efficiency was unaffected by addition of the CYT-18 protein (Fig. 15B), which binds this intron RNA tightly and is required for efficient splicing (42)(43)(44), as well as for efficient substrate cleavage by this ribozyme version (H.B. and R.R., unpublished results).…”

A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions.

“…However, the cyt4 mutants produce functional CYT-18 protein, which is active in splicing the mitochondrial large rRNA intron in vitro, and the specific activity of the CYT-18 protein in the cyt4-1 mutant is not detectably lower than in wild-type 74A (ref. 5; Qingbin Guo and A.M.L., unpublished data). These findings suggest that the CYT-4 protein does not regulate the activity of the CYT-18 protein and may instead regulate some other protein factor required for splicing, perhaps the CYT-19 protein.…”

“…IgG antibodies were purified from sera by batch adsorption to DEAE-Sephacel, chromatography through affinity columns containing TrpE and other insoluble proteins from induced Escherichia coli RR1 containing a pATH vector, and adsorption to protein A-Sepharose (5). Procedures for preparation of mitochondria and mitochondrial ribonucleoprotein (RNP) particles, SDS/polyacrylamide gel electrophoresis, immunoblotting, and glycerol gradient centrifugation were as described (5,24).…”

“…Rabbit antibodies were raised against two regions of the cyt4 ORF expressed as TrpE/ CYT-4 fusion proteins in pATH vectors, using published procedures (5,23). Plasmid pC4-3 contains a 0.4-kb Bgl II/Pst I fragment of pRAL1-cyt-4 (amino acids 115-251) cloned in pATH3, and pC4-5 contains a 1-kb Sst I/BamHI fragment of pRAL1-cyt-4 (amino acids 270-595) cloned in pATH11.…”

“…Temperature-sensitive mutants in the cyt-18 gene are defective in mitochondrial protein synthesis and in splicing the mitochondrial large rRNA intron and other group I introns in mitochondrial mRNAs (1,4). The mitochondrial tyrosyl-tRNA synthetase binds directly to the intron RNA and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro (5,6). The cyt-19-1 mutant is defective in splicing the same group I introns as the cyt-18 mutants and is presumed to be defective in a second component that functions directly in splicing, perhaps by contributing to the correct folding of the precursor RNAs or by facilitating binding of the CYT-18 protein (7,8).…”