Investigating the role and regulation of human mitochondrial poly(A) polymerase

Abstract

Polyadenylation by the mitochondrial poly(A) polymerase (mtPAP) is a crucial step of post-transcriptional modification in mammalian gene expression. In human mitochondria, polyadenylation is required for completion of seven UAA stop codons following complete processing of the major polycistronic RNA unit. Patients homozygous for a 1432A>G mutation in the PAPD1 gene, which encodes mtPAP, suffer from symptoms consistent with mitochondrial disease including autosomal-recessive spastic ataxia and optic atrophy. The principal defect of the 1432A>G mutation is short adenylate tails on mt-mRNAs. Fibroblast lines from patients harboring the 1432A>G PAPD1 mutation were established, and analysis of mitochondrial gene expression showed non-uniform dysregulation. For mt-mRNAs and translation products, there is a mix of depletion, stabilization and no effect, leading to major deficits at steady-state protein levels and of respiratory complexes. To confirm the pathological nature of the mutation, a complementation experiment was performed, which showed that expression of the WT PAPD1 gene rescued the mutant phenotype. To assess whether catalytic activity was altered in the mutant enzyme, in vitro polyadenylation assays with WT and N478D recombinant mtPAP were undertaken. The N478D mtPAP was found to generate the short oligo(A) tails as observed in vivo. In addition, the presence of the LRPPRC/SLIRP complex increased the maximal poly(A) extensions generated by both WT and mutant mtPAP. Finally, experiments were undertaken to identify factors potential interacting with mtPAP. The major interacting factor was found to be ATAD3, a protein reported to be involved with multiple mitochondrial processes involving DNA and translation machinery in the form of nucleoids or mitoribosomes respectively. In summary, these investigations provide insights into the impact and regulation of mitochondrial polyadenylation, and contribute towards unraveling the complexities of post-transcriptional maturation in human mitochondrial gene expression.