Scientific Report on the Thesis Work Carried out by Mr W. C. Wilson Whilst Funded by A

Scientific report on the thesis work carried out by Mr W. C. Wilson – whilst funded by a Pathological Society PhD Scholarship.

The human mitochondrial genome, mtDNA, encodes thirteen polypeptides, all of which are essential for the coupling of cell respiration to ATP production (OXPHOS) [1]. These proteins arise from the intra-mitochondrial translation of mt-mRNAs processed from large polycistronic precursors, which are mostly matured by the addition of short (~50nt) poly(A) tails [2, 3]. Adenylation is necessary to complete the termination codon of 7 open reading frames, but the rationale for why human mt-mRNA are polyadenylated remains unclear. Although polyadenylation of transcripts has been almost universally maintained in cells and organelles, its function varies dramatically [4-6]. In the eukaryotic cytosol polyadenylation of mRNA, when bound by poly(A) binding proteins (PABP) family members, leads to an increase in mRNA stability [6-8]. Furthermore, interactions between PABPs and factors that bind to the 5’ methylguanylate cap structure effectively circularises the transcript thereby stimulating translation initiation [9, 10]. Conversely, polyadenylation of RNA in some eubacteria can promote degradation thereby determining short-lived mRNA, it also plays a role in quality control of more stable tRNA or rRNA [11-14]. A similar lack of consistency is apparent in organelles. Yeast lack an mtPAP and no mitochondrial mRNA carries a 3’ post-transcriptional extension, whilst polyadenylation of plant and algal organellar RNA follows the eubacteria pattern and stimulates decay [15-17]. Trypanosoma mitochondria are more complicated. Short (15-20nt) oligo(A) tails are added to pre- or fully edited RNA and is necessary to promote stability, whilst U/A extensions of 200-300nt to generate longer tails designate the mRNA as ready for translation [18, 19]. It has long been known that human mt-mRNA can be polyadenylated [20], but even after numerous detailed attempts to determine its exact role [21-23], the function of this modification is still unclear. A number of different approaches by different groups have been used to address this question. These include depletion by siRNA of PAPD1 transcripts [23], obliterating poly(A) tails by targeting cytosolic PABP or poly(A) nuclease to the mitochondrion [24] and overexpression of the mitochondrial deadenylase PDE12 [22]. In each case different mt-mRNAs were analysed and different effects on stability were observed. All these methods introduced some level of biological interference but we have more recently been able to work with samples from patients who express a pathogenic mtPAP. We initially reported a mutation in PAPD1 in members of an Old Order Amish family that causes a profound form of spastic ataxia with optic atrophy [25]. We hoped that this more physiological resource could resolve the function of the poly(A) tail. 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. This is the first example of a defect of mitochondrial maturation that results in a pathogenic mitochondrial disease. The principal molecular defect of the 1432A>G mutation is short adenylate tails on mt-mRNAs (Fig. 1) as determined by mitochondrial poly(A) tail assay (MPAT). Mr Wilson modified this technique from a radiolabelled platform to one using more stable, and less toxic reagents, examples of which are shown in Figures 1 and 2. Fibroblast lines from patients harboring the 1432A>G PAPD1 mutation were established and immortalized. Following this procedure the immortalized cells lines from two patients (homozygous for the 1432A>G mutation), their unaffected sibling (heterozygous for 1432A>G) and an unrelated control were analysed. The MPAT showed clear lack of poly(A) tails in the homozygous mutant cell lines. In contrast the analysis of mitochondrial gene expression showed a non-uniform dysregulation for mitochondrial mRNAs and translation products. There are examples of reduced steady state levels, transcript stabilization resulting in elevated steady state levels or no effect. The work generated by Mr Wilson shows that these changes lead to major deficits at steady-state protein levels and also a deficit of respiratory competence (Fig. 2B and C; –LVPAPD1). It was necessary to confirm the pathological nature of the mutation. To do this all four cell lines were transduced with lentiviral particles, lines were selected for stable expression of mtPAP, and propagated to establish whether there had been any level of complementation. All the assays performed showed that expression of the WT PAPD1 gene (+LVPAPD1) rescued the mutant phenotype (Fig. 2A, B and C and further data not shown). To assess whether the catalytic activity was altered in the mutant enzyme, in vitro polyadenylation assays with WT and N478D recombinant mtPAP were undertaken. Constructs were generated to allow bacterial expression of recombinant proteins, which were then purified to homogeneity. The N478D mtPAP was found to generate short oligo(A) tails thereby recapitulating the phenotype observed in vivo. LRPPRC/SLIRP are RNA binding proteins that have been reported as important in mitochondrial gene expression. This complex was added to the poly(A) polymerase activity assays to see if there was any direct effect on poly(A) extension. 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. The importance of this observation is being investigated as part of the ongoing research in the Chrzanowska-Lightowlers lab.

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.

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