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 mitochondri al-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron tran sfer protein that is essential to respiration. However, the role of the tRN A synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein s plicing factors in this group I intron splicing reaction, we tested the hyp othesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determin ed that both the tRNA synthetase and bI4 maturase can bind directly and ind ependently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the b I4 intron bridges interactions between the two protein splicing partners. I n the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 i ntron transcribed recombinantly with flanking exons in the yeast nucleus ex hibited splicing activity. These data combined with previous genetic result s are consistent with a novel model for a ternary splicing complex (two pro tein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.