Download PPR (pentatricopeptide repeat) proteins in mammals: important aids

Biochem. J. (2008) 416, e5–e6 (Printed in Great Britain)
e5
doi:10.1042/BJ20081942
COMMENTARY
PPR (pentatricopeptide repeat) proteins in mammals: important aids to
mitochondrial gene expression
Robert N. LIGHTOWLERS1 and Zofia M. A. CHRZANOWSKA-LIGHTOWLERS
Mitochondrial Research Group, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K.
Genes encoding PPR (pentatricopeptide repeat)-containing
proteins constitute one of the largest gene families in plants. The
majority of these proteins are predicted to target organelles and
to bind to RNA. Strikingly, there is a dearth of these proteins in
mammals, although genomic searches reveal six candidates, all of
which are also predicted to target the mitochondrion. Two of these
proteins, POLRMT (the mitochondrial RNA polymerase) and
MRPS27, a mitoribosomal protein, are involved in transcription
and translation respectively. PTCD1 (pentatricopeptide repeat
domain protein 1) and PTCD3 are predicted to be involved in
the assembly of respiratory chain complexes, whereas mutations
in one other protein, LRPPRC (leucine-rich pentatricopeptide
repeat cassette), have been shown to cause defects in the levels
of cytochrome c oxidase, the terminal member of the respiratory
chain. In this issue of the Biochemical Journal, Xu et al. turn
their attention to the remaining candidate, PTCD2. Depletion in
a mouse model led to deficiencies of the third complex of the
respiratory chain that caused profound ultrastructural changes
in the heart. The exact molecular function of PTCD2 remains
unclear, but depletion leads to an apparent lack of processing of
the mitochondrial transcript encoding apocytochrome b, a critical
member of complex III. These data are consistent with PTCD2
playing an important role in the post-transcriptional expression of
the mitochondrial genome.
INTRODUCTION
RNA species that are generated require a number of processes to
be completed to produce mature transcripts, including separation
of polycistrons into individual RNA units, removal of intronic
sequences, RNA editing and protection against degradation before
loading on to ribosomes for translation. It is clear that the PPR
proteins have RNA-binding activities and demonstrate sequence
specificity, but at what stages of an RNA molecule’s life-cycle
might the PPR proteins interact with their substrate? RNA editing
is a common post-transcriptional mechanism in plant organelles,
and also occurs in protist mitochondria, but is vanishingly rare
in mammalian mitochondria. This led to an initial hypothesis
that PPR proteins played crucial roles in RNA editing. Indeed,
this was subsequently confirmed experimentally. One particular
mutational analysis of a PPR protein, CRR4, resulted in the loss
of editing of a crucial translation initiation codon of a chloroplast
transcript encoding a member of the NA(P)DH dehydrogenase
complex (for a review, see [4]). Numerous other examples now
also abound [4].
Almost 10 years ago, Peeters and Small searched the part of
the Arabidopsis thaliana genome that had then been sequenced
(70 %), specifically looking for the genes encoding proteins that
were predicted to be targeted to mitochondria and chloroplasts.
They got much more than they bargained for: a previously
unreported unique gene family that comprised almost 200 genes.
Subsequent detailed analysis of the entire A. thaliana genome
revealed this family to contain 450 independent genes separated
into two subfamilies and four subclasses [1,2].
WHAT WERE THESE PROTEINS?
These transpired to be an extensive gene family encoding PPR
(pentatricopeptide repeat) proteins that are characterized by a
structural motif of a degenerate 35-amino-acid sequence, which
appears as tandem repeats. Structural predictions of specific PPR
proteins were suggestive of RNA binding, and subsequent experimental evidence has confirmed RNA substrate selectivity for
numerous members of this family. Then, there followed another
surprise: this vast gene family appeared to be almost exclusive to
plants, with virtually no representatives in prokaryote genomes,
approx. 20 in protists and only a handful of candidates in other
non-plant eukaryotes. However, in an analogous manner to that
in plants, the meagre six PPR proteins encoded by the human
genome are all predicted to be mitochondrial [3].
WHAT IS THE PHYSIOLOGICAL FUNCTION OF
THESE PPR PROTEINS?
To aid in this prediction, it is important to remember that both
chloroplasts and mitochondria have their own DNA (cDNA
and mtDNA respectively), which is transcribed and the mRNA
translated within the relevant organelle. The primary organellar
1
Key words: gene expression, mitochondrion, pentatricopeptide
repeat protein, post-transcriptional regulation, respiratory chain,
RNA.
ARE ALL PPR PROTEINS INVOLVED ONLY IN EDITING?
This seems unlikely, as it would make the mammalian proteins
redundant. Hence, although this initial link between PPR proteins
and the RNA editing hypothesis was seductive, it has become clear
that not only do PPR proteins function as site-specific markers in
RNA editing, but also at all other post-transcriptional stages of
mRNA expression. Various plant PPR proteins have now been
characterized and demonstrate functions beyond editing; in RNA
splicing, enhancing transcript stability and also as translational
activators through interactions with untranslated regions [4].
Since neither editing nor splicing are features of human mtRNA,
and untranslated regions are uncommon, the roles played by
human PPR proteins must be more subtle, but may potentially
affect translation.
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
e6
R. N. Lightowlers and Z. M. A. Chrzanowska-Lightowlers
WHAT IS KNOWN ABOUT HUMAN PPR PROTEINS?
As mentioned earlier, even after analysis of the entire human
genome, these appear to be limited to only six members. The first
of these to be identified came via an elegant use of integrative
genomics in a landmark paper revealing how integration of
datasets on RNA expression, organellar proteomics and genomic
maps could provide an approach that has the potential to expedite
disease-gene discovery [5]. This technique was validated as
mutations in the gene encoding this PPR protein, LRPPRC
(leucine-rich pentatricopeptide repeat cassette), were shown to
cause a rare form of inherited COX (cytochrome c oxidase)
deficiency that was termed ‘Leigh Syndrome French-Canadian
type’ (or LSFC), as it was found in the population of the SaguenayLac St-Jean region of Quebec [5].
In the first of their studies on human PPR proteins, Xu et al.
[6] reported that at least a subset of LRPPRC protein is naturally
imported into mitochondria. Furthermore, in cell lines derived
from the COX-defective LSFC patients, the steady-state levels of
mature MTCO1–3 transcripts were reported to be depleted. Consistent with this observation, in vivo synthesis of COX1 and COX3
proteins were also reportedly low [6]. From these data, it is difficult to determine the exact role of LRPPRC, but if it is involved
in processing of the primary RNA unit or in stabilizing mature
transcripts it can only have a very limited sequence selectivity,
unlike many of the plant PPR proteins that have been assessed.
In this issue of the Biochemical Journal, Xu et al. [7] turn
their attention to a second mammalian PPR protein, PTCD2
(pentatricopeptide repeat domain protein 2). Using gene-trap
technology, they have engineered a homozygous disruption of
the PTCD2-encoding gene in mice. In mammals, all of the
polypeptides encoded by the mitochondrial genome are members
of the five complexes that couple respiration to ATP synthesis. To
identify whether mitochondrial homoeostasis had been affected
as a consequence of PTCD2 disruption, the authors analysed
the respiratory chain enzyme activities in various tissues. The
data revealed that there were defects in complex III (ubiquinol:
cytochrome c oxidoreductase) activity in the heart, and an even
greater defect of complexes I plus III activity in the liver and
heart of the homozygote. More detailed analyses of the heart
by light microscopy showed severe ultrastructural abnormalities,
particularly in the myocardium. Blue native-gel electrophoresis
followed by Western blotting was used to assess the steady-state
level of fully assembled complex III, which had appeared to be
biochemically defective. Three different antibodies were used to
probe the status of complex III, and the authors conclude that
levels of the native complex were reduced. Using the data derived
from Northern blots, the authors conclude that there is depletion
of the matured MTCYB transcript with a concomitant increase in
a larger precursor, with the latter including MTND5, presumably
together with the antisense of MTND6 and MTTE.
COULD THE ROLE OF PTCD2 BE TO FACILITATE THE PROCESSING
OF THIS INTERMEDIATE PRECURSOR RNA?
Since the report of the entire human mitochondrial genome
in 1981 [8], it has become clear that mammalian mtDNA
Received 25 September 2008; accepted 26 September 2008
Published on the Internet 28 October 2008, doi:10.1042/BJ20081942
c The Authors Journal compilation c 2008 Biochemical Society
is transcribed from both strands from promoters within the
major non-coding region. This generates large polycistronic RNA
species, and it has been generally accepted that the large primary
transcript is aided in its processing by the spontaneous folding of
the cloverleaf mitochondrial tRNA structures that punctuate it [3].
These mitochondrial tRNAs are then processed by a mitochondrial
RNase P, and potentially by an RNase Z type activity. This
mechanism is highly likely to occur, but cannot account for the
full processing of the entire primary polycistronic unit as there
are numerous precursors that are not punctuated by tRNAs. The
authors, however, identified the large precursor RNA described
above in the −/− homozygote that does contain an antisense
tRNA, which may potentially recruit the RNases in a similar
fashion. The apparent loss of mature MTND5, which remained
locked in the large precursor, is also intriguing.
COULD THIS UNPROCESSED TRANSCRIPT STILL BE LOADED ON
TO MITORIBOSOMES AND TRANSLATED IRRESPECTIVE THAT IT
HAD BEEN INCOMPLETELY PROCESSED?
This is certainly a compelling question and it would be interesting
to assess the rate of synthesis of the mitochondrial gene products
after the PTCD2 disruption. These are experiments for the future,
which could perhaps be performed in a standard human cell line
with a simple siRNA (small interfering RNA)-mediated downregulation of PTCD2. All will undoubtedly help us to understand
better the molecular mechanisms governing mitochondrial posttranscriptional gene expression.
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