The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown to be defective for rps12 trans-splicing. The observations that PPR4 is associated in vivo with rps12-intron 1 and that it is also required for its splicing demonstrate that PPR4 is an rps12 trans-splicing factor. These findings add trans-splicing to the list of RNA-related functions associated with PPR proteins and suggest that plastid group II trans-splicing is performed by different machineries in vascular plants and algae.
Comparative genomics has provided evidence for numerous conserved protein domains whose functions remain unknown. We identified a protein harboring ''domain of unknown function 860'' (DUF860) as a component of group II intron ribonucleoprotein particles in maize chloroplasts. This protein, assigned the name WTF1 (''what's this factor?''), coimmunoprecipitates from chloroplast extract with group II intron RNAs, is required for the splicing of the introns with which it associates, and promotes splicing in the context of a heterodimer with the RNase III-domain protein RNC1. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. DUF860 is found only in plants, where it is represented in a protein family comprising 14 orthologous groups in angiosperms. Most members of the DUF860 family are predicted to localize to chloroplasts or mitochondria, suggesting that proteins with this domain have multiple roles in RNA metabolism in both organelles. These findings add to emerging evidence that the coevolution of nuclear and organellar genomes spurred the evolution of diverse noncanonical RNA-binding motifs that perform organelle-specific functions.DUF860 ͉ mitochondria ͉ plastid T he evolution of mitochondria and chloroplasts from bacterial endosymbionts was accompanied by large scale transfer of genes to the nucleus (1). Accordingly, many organellar proteins are encoded by nuclear genes of bacterial ancestry that retain their ancestral function. However, during the long coevolution of mitochondria and chloroplasts with their host cell, both organelles acquired features that are not typical of their bacterial ancestors. The origin of the genes that confer such traits is only beginning to be elucidated.The complex RNA metabolism characteristic of plant mitochondria and chloroplasts provides striking examples of acquired, nonprocaryotic traits. For example, both organellar genomes are rich in introns, RNAs in both organelles are modified by RNA editing, and posttranscriptional events have the predominant role in determining gene product abundance (2, 3-5). Many proteins that participate in such processes were not derived from the endosymbiont, but rather emerged in the context of nuclear-organellar coevolution. For example, the pentatricopeptide repeat (PPR) protein family is found only in eucaryotes, where it has been implicated in RNA-related processes in mitochondria and chloroplasts (6). Current data argue that PPR proteins generally function as RNA-interaction platforms, but they appear to be derived from the tetratricopeptide repeat (TPR) motif, a more ancient motif that binds protein ligands.Here, we present evidence that a previously uncharacterized protein family defined by ''domain of unknown function 860'' (DUF860) fits this general paradigm. We show that the DUF860 protein WTF1 (''what's this factor?'') is required for the splicing of group II introns in chloroplasts, that it associates in vivo with its genetically-defined RNA ligands, and tha...
SUMMARYHigh-copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype-driven forward genetics has been hampered by the difficulty of identifying the specific insertions responsible for phenotypes of interest. We describe a new method that can substantially increase the throughput of linking a disrupted gene to a known phenotype in high-copy Mutator (Mu) transposon lines in maize. The approach uses the Illumina platform to obtain sequences flanking Mu elements in pooled, barcoded DNA samples. Insertion sites are compared among individuals of suitable genotype to identify those that are linked to the mutation of interest. DNA is prepared for sequencing by mechanical shearing, adapter ligation, and selection of DNA fragments harboring Mu flanking sequences by hybridization to a biotinylated oligonucleotide corresponding to the Mu terminal inverted repeat. This method yields dense clusters of sequence reads that tile approximately 400 bp flanking each side of each heritable insertion. The utility of the approach is demonstrated by identifying the causal insertions in four genes whose disruption blocks chloroplast biogenesis at various steps: thylakoid protein targeting (cpSecE), chloroplast gene expression (polynucleotide phosphorylase and PTAC12), and prosthetic group attachment (HCF208/CCB2). This method adds to the tools available for phenotype-driven Mu tagging in maize, and could be adapted for use with other high-copy transposons. A by-product of the approach is the identification of numerous heritable insertions that are unrelated to the targeted phenotype, which can contribute to community insertion resources.
Chloroplast genomes in land plants harbor ;20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1-and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.
Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are related to the tetratricopeptide repeat (TPR). Most characterized PPR proteins mediate specific post-transcriptional steps in gene expression in mitochondria or chloroplasts. However, little is known about the structure of PPR proteins or the biochemical mechanisms through which they act. Here we establish features of PPR protein structure and nucleic acid binding activity through in vitro experiments with PPR5, which binds and stabilizes a chloroplast tRNA precursor harboring a group II intron. Recombinant PPR5 was shown to be monomeric by analytical ultracentrifugation and gel filtration. Circular dichroism spectroscopy showed that PPR5 has a high content of a helices, as predicted from the similarity between PPR and TPR motifs. PPR5 and another PPR protein, CRP1, bind with high affinity to single-stranded RNA, but bind poorly to single-stranded DNA or to double-stranded RNA or DNA. A specific PPR5 binding site was identified within its group II intron ligand. The minimal site spans ;45 nucleotides, encompasses two group II intron functional motifs, and overlaps the terminus of an in vivo RNA decay product. These results suggest mechanisms by which PPR5 influences both RNA stability and splicing.
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