* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download PPR (pentatricopeptide repeat) proteins in mammals: important aids
Deoxyribozyme wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Human genome wikipedia , lookup
Messenger RNA wikipedia , lookup
Genome evolution wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Gene expression profiling wikipedia , lookup
Nucleic acid tertiary structure wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
Polyadenylation wikipedia , lookup
RNA interference wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Protein moonlighting wikipedia , lookup
Mir-92 microRNA precursor family wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
RNA silencing wikipedia , lookup
History of RNA biology wikipedia , lookup
Epitranscriptome wikipedia , lookup
Non-coding RNA wikipedia , lookup
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. REFERENCES 1 Small, I. D. and Peeters, N. (2000) The PPR motif – a TPR-related motif prevalent in plant organellar proteins. Trends Biochem. Sci. 25, 46–47 2 Lurin, C., Andrés, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyère, C., Caboche, M., Debast, C., Gualberto, J., Hoffmann, B. et al. (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103 3 Ojala, D., Montoya, J. and Attardi, G. (1981) tRNA punctuation model of RNA processing in human mitochondria. Nat. Genet. 290, 470–474 4 Delannoy, E., Stanley, W. A., Bond, C. S. and Small, I. D. (2007) Pentatricopeptide repeat (PPR) proteins as sequence-specificity factors in post-transcriptional processes in organelles. Biochem. Soc. Trans. 35, 1643–1647 5 Mootha, V. K., Lepage, P., Miller, K., Bunkenborg, J., Reich, M., Hjerrild, M., Delmonte, T., Villeneuve, A., Sladek, R., Xu, F. et al. (2003) Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics. Proc. Natl. Acad. Sci. U.S.A. 100, 605–610 6 Xu, F., Morin, C., Mitchell, G., Ackerley, C. and Robinson, B. H. (2004) The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA. Biochem. J. 382, 331–336 7 Xu, F., Ackerley, C., Maj, M. C., Addis, J. B. L., Levandovskiy, V., Lee, J., MacKay, N., Cameron, J. M. and Robinson, B. H. (2008) Disruption of a mitochondrial RNA-binding protein gene results in decreased cytochrome b expression and a marked reduction in ubiquinol–cytochrome c reductase activity in mouse heart mitochondria. Biochem. J. 416, 15–26 8 Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F. et al. (1981) Sequence and organization of the human mitochondrial genome. Nat. Genet. 290, 457–465