The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity.
The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.
Rho
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Department of Biology and Biochemistry, University of Houston, Texas 77204-5513, USA.
Martinis
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SA
D001483:Base Sequence; D003573:Cytochrome b Group / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D007438:Introns / Q000502:physiology*; D007935:Leucine-tRNA Ligase / Q000378:metabolism*; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
D003573:Cytochrome b Group; D005656:Fungal Proteins; D046911:Macromolecular Substances; D016337:RNA, Catalytic; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C066564:aI2 protein, S cerevisiae; D012194:RNA-Directed DNA Polymerase; D007935:Leucine-tRNA Ligase