Two Related RNA-editing Proteins Target the Same Sites in Mitochondria of Arabidopsis thaliana

The facilitators for specific cytosine-to-uridine RNA-editing events in plant mitochondria and plastids are pentatricopeptide repeat (PPR)-containing proteins with specific additional C-terminal domains. Here we report the related PPR proteins mitochondrial editing factor 8 (MEF8) and MEF8S with only five such repeats each to be both involved in RNA editing at the same two sites in mitochondria of Arabidopsis thaliana. Mutants of MEF8 show diminished editing in leaves but not in pollen, whereas mutants of the related protein MEF8S show reduced RNA editing in pollen but not in leaves. Overexpressed MEF8 or MEF8S both increase editing at the two target sites in a mef8 mutant. Double mutants of MEF8 and MEF8S are not viable although both identified target sites are in mRNAs for nonessential proteins. This suggests that MEF8 and MEF8S may have other essential functions beyond these two editing sites in complex I mRNAs.     

A trypanosomal pentatricopeptide repeat protein stabilizes the mitochondrial mRNAs of cytochrome oxidase subunits 1 and 2

The pentatricopeptide repeat (PPR) protein family consists of organellar proteins predicted to bind to specific RNA sequences. Plants have hundreds of distinct PPR proteins, whereas other eukaryotes generally have many fewer. The genome of the parasitic protozoon Trypanosoma brucei is predicted to encode more than 30 different PPR proteins, which is an extraordinarily high number for a nonplant organism. Here we report the characterization T. brucei PPR9 (TbPPR9). Epitope tagging shows that the protein is exclusively mitochondrially localized. Interestingly, while in induced RNA interference cell lines TbPPR9 is efficiently downregulated, the level of its mRNA is not affected. Ablation of TbPPR9 selectively abolishes oxidative but not mitochondrial substrate-level phosphorylation. The molecular basis of this phenotype is the fact that TbPPR9 is required for the stability of the cytochrome oxidase subunit 1 (COX1) and COX2 mRNAs. This is supported by the observation that ablation of TbPPR9 destabilizes the COX complex but not the cytochrome bc1 or the ATP synthase complex. Moreover, it was shown by blue native gel electrophoresis that TbPPR9 is present in a large complex of unknown composition.     

MRPS27 is a pentatricopeptide repeat domain protein required for the translation of mitochondrially encoded proteins

Mammalian pentatricopeptide repeat domain (PPR) proteins are involved in regulation of mitochondrial RNA metabolism and translation and are required for mitochondrial function. We investigated an uncharacterised PPR protein, the supernumerary mitochondrial ribosomal protein of the small subunit 27 (MRPS27), and show that it associates with the 12S rRNA and tRNA^Glu, however it does not affect their abundance. We found that MRPS27 is not required for mitochondrial RNA processing or the stability of the small ribosomal subunit. However, MRPS27 is required for mitochondrial protein synthesis and its knockdown causes decreased abundance in respiratory complexes and cytochrome c oxidase activity.

Structured summary of protein interactions

MRPS27 and MRPS15 colocalize by cosedimentation through density gradient (View Interaction)     

LRP130, a pentatricopeptide motif protein with a noncanonical RNA-binding domain,is bound in vivo to mitochondrial and nuclear RNAs

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.     

The potential for manipulating RNA with pentatricopeptide repeat proteins

The pentatricopeptide repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence‐specific RNA‐binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2‐30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA‐binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR‐based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.     

A RESTORER OF FERTILITY-like PPR gene is required for 5'-end processing of the nad4 mRNA in mitochondria of Arabidopsis thaliana

Processing of 5'-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5' leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5'-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex. This nuclear gene, At1g12700, that we designate RNA PROCESSING FACTOR 1 (RPF1), encodes a pentatricopeptide repeat (PPR) protein of the P-class containing canonical PPR-repeats. RPF1 belongs to a subgroup of PPR proteins, which includes the RESTORER OF FERTILITY (RF) gene products restoring cytoplasmic male sterility (CMS) in various plant species. CMS is a mitochondrially inherited trait caused by the expression of aberrant, chimeric genes, which has not been observed in the predominantly inbreeding species Arabidopsis thaliana. The here reported results are a further step towards the characterization of the plant mitochondrial RNA processing machinery and provide additional insights into the function of RF-like PPR proteins.     

RNA-binding proteins of mammalian mitochondria

A UV-cross-linking assay was used to identify RNA-binding proteins in mammalian mitochondria. A number of these proteins were detected ranging in molecular mass from 15 to 120 kDa. All of the mRNA-binding activities were localized to the matrix except for two proteins which are primarily associated with the inner membrane. None of the polypeptides is specific for binding mitochondrial mRNAs since all bound mRNAs from other sources with comparable efficiency. Some preference for binding mRNA over tRNA or homoribopolymers was observed with several of the proteins. A protein with characteristic pentatricopeptide repeat motifs found in many RNA binding proteins was identified associated with the small subunit of the mitochondrial ribosome.