Pentatricopeptide Repeat Proteins in Trypanosoma brucei Function in Mitochondrial Ribosomes

Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, CH-1700, Fribourg, Switzerland.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2007; 27(19):6876-88. DOI: 10.1128/MCB.00708-07
Source: PubMed


The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have
400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been
studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic
protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A comparative analysis shows that
seven out of eight selected PPR proteins are mitochondrially localized and essential for oxidative phosphorylation. Six of
these are required for the stabilization of mitochondrial rRNAs and, like ribosomes, are associated with the mitochondrial
membranes. Furthermore, one of the PPR proteins copurifies with the large subunit rRNA. Finally, ablation of all of the PPR
proteins that were tested induces degradation of the other PPR proteins, indicating that they function in concert. Our results
show that a significant number of trypanosomal PPR proteins are individually essential for the maintenance and/or biogenesis
of mitochondrial rRNAs.

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    • "Downloaded from Published October 19 , 2015 Trypanosome development • Domingo - Sananes et al . RNA polymerase and tetracycline repressor protein ( Engstler and Boshart , 2004 ) . RNA constructs were based on the pALC 14 plasmid ( Pusnik et al . , 2007 ) . Transfection and cell maintenance in HMI - 9 me - dium containing 20% FCS ( Hirumi and Hirumi , 1989 ) was performed as previously described ( Szöőr et al . , 2013 ) ."
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    ABSTRACT: The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, proliferation, and transmission of these parasites. One of these, the differentiation of growth-arrested stumpy forms in the mammalian blood into insect-stage procyclic forms, can be induced synchronously in vitro with cis-aconitate. Here, we show that this transition is an irreversible bistable switch, and we map the point of commitment to differentiation after exposure to cis-aconitate. This irreversibility implies that positive feedback mechanisms operate to allow commitment (i.e., the establishment of "memory" of exposure to the differentiation signal). Using the reversible translational inhibitor cycloheximide, we show that this signal memory requires new protein synthesis. We further performed stable isotope labeling by amino acids in cell culture to analyze synchronized parasite populations, establishing the protein and phosphorylation profile of parasites pre- and postcommitment, thereby defining the "commitment proteome." Functional interrogation of this data set identified Nek-related kinase as the first-discovered protein kinase controlling the initiation of differentiation to procyclic forms.
    The Journal of Cell Biology 10/2015; 211(6). DOI:10.1083/jcb.201506114 · 9.83 Impact Factor
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    • "In T. brucei, more than 40 PPRs, including those associated with the KPAP1 complex [78] [79] and mitochondrial ribosomes [97] have been annotated (reviewed in Ref. [98]). At least some ribosome-associated PPRs are essential for rRNA biogenesis or stability, as evidenced by the loss of either 9S or 12S rRNAs in respective RNAi knockdowns [99]. In agreement with their proposed function, knockdown of KPAF1 and 2 led to selective elimination of the A/U-tailed mRNAs while leaving short A-tailed mRNAs unaffected [79]. "
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    ABSTRACT: Mitochondrial mRNA editing in trypanosomes is a posttranscriptional processing pathway thereby uridine residues (Us) are inserted into, or deleted from, messenger RNA precursors. By correcting frameshifts, introducing start and stop codons, and often adding most of the coding sequence, editing restores open reading frames for mitochondrially-encoded mRNAs. There can be hundreds of editing events in a single pre-mRNA, typically spaced by few nucleotides, with U-insertions outnumbering U-deletions by approximately 10-fold. The mitochondrial genome is composed of ∼50 maxicircles and thousands of minicircles. Catenated maxi- and mini-circles are packed into a dense structure called the kinetoplast; maxicircles yield rRNA and mRNA precursors while guide RNAs (gRNAs) are produced predominantly from minicircles, although varying numbers of maxicircle-encoded gRNAs have been identified in kinetoplastids species. Guide RNAs specify positions and the numbers of inserted or deleted Us by hybridizing to pre-mRNA and forming series of mismatches. These 50-60 nucleotide (nt) molecules are 3' uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Editing reactions of mRNA cleavage, U-insertion or deletion, and ligation are catalyzed by the RNA editing core complex (RECC). To function in mitochondrial translation, pre-mRNAs must further undergo post-editing 3' modification by polyadenylation/ uridylation. Recent studies revealed a highly compound nature of mRNA editing and polyadenylation complexes and their interactions with the translational machinery. Here we focus on mechanisms of RNA editing and its functional coupling with pre- and post-editing 3' mRNA modification and gRNA maturation pathways.
    Biochimie 01/2014; 100C(1). DOI:10.1016/j.biochi.2014.01.003 · 2.96 Impact Factor
    • "Until now, most MRP studies have focused on yeast, bacteria and mammals, such as rat and bovine. There are only a few reports about MRPs in protozoa and cestodes, such as Trypanosoma brucei (Pusnik et al., 2007; Zikova et al., 2008) and Echinococcus multilocularis (Mathis et al., 2005), and there are no reports regarding MRPs in parasitic nematodes , including T. spiralis. Because most MRPs are synthesized by cytoplasmic ribosomes and are important for mitochondrial protein synthesis, the inhibition of MRP synthesis in ribosomes by antibiotics might be a new way to control the parasites. "
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    ABSTRACT: A cDNA encoding mitochondrial ribosomal protein (MRP)-L28 was isolated and cloned from Trichinella spiralis, an economically important parasitic nematode. The predicted TsMRP-L28 protein consists of 276 amino acids. Phylogenetic analysis revealed that TsMRP-L28 was closely related to Caenorhabditis elegans mitochondrial ribosomal protein L28. The TsMRP-L28 transcript was expressed in newborn larvae, muscle larvae and male and female adult worms. Western blot showed that TsMRP-L28 was expressed in muscle larvae and adult worms. Immuno-localization revealed that TsMRP-L28 was ubiquitously distributed in newborn larvae, muscle larvae and adult worms, and that TsMRP-L28 was enriched in cells with higher protein synthesis activity, such as in newborn larvae and the cytoplasm of different developmental stages of embryos. These data suggest that TsMRP-L28 is required for the early development of T. spiralis.
    Gene 10/2012; 512(2). DOI:10.1016/j.gene.2012.10.010 · 2.14 Impact Factor
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