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 preor 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 posttranscriptional 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
A
Poly(A) Oligo(A)
B
C
Figure 1. Fluorescent MPAT. Fluorescently labeled
samples from two homozygous PAPD1 mutant RNA samples,
one heterozygote carrying the 1432A>G PAPD1 mutation, and
a PCR control were separated on a 10% polyacrylamide 8.2M
urea denaturing gel. Visualized on Storm PhosphorImager.
mutation is short adenylate tails on mtmRNAs (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
Figure 2. Suppression of PAPD1 mutation phenotype by
expression of wildtype PAPD1. Analyses before (-LVPAPD1) or
after (+LVPAPD1) lentiviral transduction with wildtype mitochondrial
poly(A) polymerase. MPAT as Fig. 1 (A) with lane profiles shows
restored ratio of oligo to poly(A) tail lengths. Northern (B) and western
(C) analyses are of mitochondrially encoded (COX1, COX2, ND1, ND3,
ATP8, RNA14) RNA or protein respectively. Cytosolic (18S rRNA) or
nuclear encoded but mitochondrially located (SDHA, NDUFA9,
NDUFB8) controls were analysed as was the level of mitochondrial
poly(A) polymerase protein.
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
Figure 3. Poly(A) polymerase activity
assays. Assays were performed with wildtype or
mutant mtPAP and RNA template corresponding to
the 3’ terminus of a mitochondrial transcript
(RNA14) or with an oligoA8 tail (RNA14-A8).
recapitulating
the
phenotype
observed in
vivo. LRPPRC/SLIRP are RNA binding proteins that
have been reported as important in mitochondrial gene
Figure 4. Figure 3. Poly(A) polymerase
expression. This complex was added to the poly(A)
wildtype or mutant mtPAP and RNA template
corresponding to the 3’ terminus of a mitochondrial
transcript (RNA14) or with an oligoA8 tail (RNA14A8). Assays were performed with or without the
addition of the RNA binding proteins LRPPRC and
SLIRP.
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
activity assays. Assays were performed with
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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