Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns

Abstract

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic α-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted α-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 α-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA Tyr, but not for TyrRS activity with Escherichia coli tRNA Tyr, implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.