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.

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 alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-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 alpha-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.

Recombinant adeno-associated virus-mediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse.

The central nervous system (CNS) is a predominant site of involvement in several lysosomal storage diseases (LSDs); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. The mucopolysaccharidosis type VII (MPS VII) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and leptomeningeal cells. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) vector is capable of reversing the progression of storage lesions within the CNS. Adult MPS VII mice received intracerebral injections of 4 X 10(7) infectious units of a rAAV vector carrying the murine beta-glucuronidase (gus-s(a)) cDNA under the transcriptional direction of the cytomegalovirus immediate-early promoter and enhancer. By 1 month after vector administration, transgene-derived beta-glucuronidase was present surrounding the injection site. Enzyme levels were between 50 and 240% of that found in wild-type mice. This level of beta-glucuronidase activity was sufficient to reduce the degree of lysosomal storage. Moreover, the reduction in storage was maintained for at least 3 months post-rAAV administration. These data demonstrate that rAAV vectors can transduce the diseased CNS of MPS VII mice and mediate levels of transgene expression necessary for a therapeutic response. Thus, rAAV vectors are potential tools in the treatment of the mucopolysaccharidoses and other lysosomal storage diseases.