The bl4 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 (bl4) 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 bl4 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 bl4 maturase interact directly with the bl4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bl4 maturase can bind directly and independently via RNA-protein interactions to the large bl4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bl4 intron bridges interactions between the two protein splicing partners. In the presence of either the bl4 maturase or the Leu-tRNA synthetase, bl4 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 bl4 intron and facilitate its self-splicing activity.