PhD student, The University of Western Australia
Pentatricopeptide Repeat (PPR) Proteins are modular RNA-binding proteins with repeated 35-amino acid sequence motifs that control mitochondrial and plastid gene expression. They are being considered as a potential tool for manipulating gene expression in plants because they can recognise a wide range of different RNA sequences and the molecular basis for this sequence recognition is partially known and understood, allowing their specificity to be deliberately altered. Each PPR motif recognises and binds a single nucleotide with a 1:1 correspondence between contiguous protein motifs and RNA bases in the target RNA. This recognition is determined primarily by hydrogen bonding patterns between the RNA base and two amino acid side chains at specific positions in each PPR motif. Modifying these amino acids should alter target specificity. The project should expand our knowledge of RNA specificity and binding by PPR proteins in plant mitochondria and contribute to the ultimate goal of developing specific RNA targeting tools using PPR proteins that can be aimed at desired targets.
Abstract: The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA-binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA-protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.
Pub.: 24 Aug '12, Pinned: 22 Aug '17
Abstract: Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants, with more than 400 members in most species. Over the past decade, much has been learned about the molecular functions of these proteins, where they act in the cell, and what physiological roles they play during plant growth and development. A typical PPR protein is targeted to mitochondria or chloroplasts, binds one or several organellar transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. Their combined action has profound effects on organelle biogenesis and function and, consequently, on photosynthesis, respiration, plant development, and environmental responses. Recent breakthroughs in understanding how PPR proteins recognize RNA sequences through modular base-specific contacts will help match proteins to potential binding sites and provide a pathway toward designing synthetic RNA-binding proteins aimed at desired targets.
Pub.: 30 Jan '14, Pinned: 22 Aug '17
Abstract: 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.
Pub.: 30 Jan '14, Pinned: 22 Aug '17
Abstract: We report the partial complementation and subsequent comparative molecular analysis of two non-viable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript, and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA 'footprint' located on the 5' half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence-specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.
Pub.: 25 Dec '16, Pinned: 22 Aug '17