Leucyl-tRNA Synthetase-dependent and -independent Activation of a Group I Intron

Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.Although the aminoacyl-tRNA synthetases (aaRSs)⁶ are best known for their role in protein synthesis, many have functionally expanded and are essential to a wide range of other cellular activities that are unrelated to tRNA aminoacylation (1). The class I aaRSs, leucyl- (LeuRS or NAM2) and tyrosyl-tRNA synthetase (TyrRS or CYT-18) are required for RNA splicing of cognate group I introns in the mitochondria of certain lower eukaryotes (2). In yeast, processing of two related group I introns called bI4 and aI4α (Fig. 1) from the cob and cox1α genes, respectively, require yeast mitochondrial LeuRS (3, 4). Likewise, expression of Neurospora crassa mitochondrial genes, such as those for the large ribosomal RNA, is dependent on TyrRS for excising group I introns (5).Open in a separate windowFIGURE 1.Predicted secondary structures of the bI4 and aI4α group I introns. The secondary structure of the canonical core was based on previous predictions (19). Solid bold lines indicate linear connectivities of the nucleic acid strand with arrowheads oriented in the 5′ to 3′ direction. The dashed lines represent putative tertiary interactions. Dotted lines with numbers identify insertions where secondary structures were ambiguous. Arrows in the P1 and P9 domain show splice sites, whereas boxed nucleotides are paired regions.LeuRS facilitates RNA splicing in concert with a bI4 maturase that is encoded within the bI4 intron. Genetic investigations showed that an inactivated bI4 maturase resulting in deficient splicing activity of the bI4 and aI4α group I introns can be rescued by a suppressor mutation of LeuRS to restore mitochondrial respiration (4, 6). In addition, the splicing defect can be compensated by a mutant aI4α DNA endonuclease that is closely related to the bI4 maturase (7, 8).Previously, we used intracellular three-hybrid assays to demonstrate that LeuRS and bI4 maturase can independently bind to the bI4 intron and stimulate RNA splicing activity in the non-physiological yeast nucleus compartment (9). RNA-dependent two-hybrid assays also supported that the bI4 intron could simultaneously bind both the bI4 maturase and LeuRS. In this case, the RNA was co-expressed with LeuRS and bI4 maturase that was fused to either LexA or B42 to generate a two-hybrid response. This suggested that the bI4 intron was bridging these two protein splicing factors. In either the RNA-dependent two-hybrid or three-hybrid assays, bI4 intron splicing occurred only in the presence of LeuRS or bI4 maturase or both.We hypothesized that the bI4 maturase and LeuRS bind to distinct sites of the bI4 intron to form a ternary complex and promote efficient splicing activity. However, the functional basis of the collaboration between these two splicing cofactors or how either of them promotes RNA splicing remains unclear.We sought to characterize the respective splicing roles of the bI4 maturase and LeuRS via biochemical investigations. Previous attempts to develop an in vitro splicing assay for the bI4 intron or its closely related aI4α intron have failed (10, 11). It was hypothesized that the long length of the bI4 intron (∼1600 nucleotides) and its highly A:U-rich content (∼80%) hindered RNA folding in vitro as well as stabilization of its competent structure.Efforts to produce an active form of the bI4 intron have relied on building chimeric group I introns by interchanging RNA domains with the more stable Tetrahymena thermophila group I intron (11). Based on these results, it was proposed that the catalytic core of the bI4 group I intron was inherently defective (11). In this case, the group I intron would be expected to be completely dependent on its protein splicing factors similar to the bI3 intron that relies on the bI3 maturase and Mrs1 for activity (12). Thus, it was hypothesized that the bI4 maturase and/or LeuRS splicing factors aided the bI4 group I intron by targeting its core region to compensate for these deficiencies.We focused our efforts on re-designing the bI4 intron to develop a minimized molecule that might be competent for splicing. Because both the bI4 and aI4α group I introns rely on the bI4 maturase and LeuRS for their splicing activity, we compared their secondary structures to identify and eliminate peripheral regions outside of their catalytic cores. A small active derivative of the bI4 intron, comprised of just 380 nucleotides primarily from the canonical core, was generated. Thus, we show that, in and of itself, the canonical core of this group I intron is competent for splicing. Both the bI4 maturase and LeuRS enhance the splicing activity of the minimized bI4 intron. However, it is possible that protein-dependent splicing of the bI4 intron represents an intermediate evolutionary step in which the RNA activity is becoming increasingly dependent on its protein splicing factors.