Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.
Open Access
- 1 May 1989
- journal article
- research article
- Published by Taylor & Francis in Molecular and Cellular Biology
- Vol. 9 (5), 2089-2104
- https://doi.org/10.1128/mcb.9.5.2089
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). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.This publication has 38 references indexed in Scilit:
- A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereofCell, 1987
- Protein-dependent splicing of a group I intron in ribonucleoprotein particles and soluble fractionsCell, 1986
- Enzymatic activity of the conserved core of a group I self-splicing intronNature, 1986
- Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interfaceBiochemistry, 1985
- RNA splicing in Neurospora mitochondriaJournal of Molecular Biology, 1985
- RNA splicing in neurospora mitochondria: Self-splicing of a mitochondrial intron in vitroCell, 1984
- Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-SepharoseImmunochemistry, 1978
- High resolution two-dimensional electrophoresis of basic as well as acidic proteinsCell, 1977
- Affinity Elution as a Purification Method for Aminoacyl‐tRNA SynthetasesEuropean Journal of Biochemistry, 1973
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970