Rosalind Williams-Carrier

RNA immunoprecipitation and microarray analysis show a chloroplast Pentatricopeptide repeat protein to be associated with the 5' region of mRNAs whose translation it activates.

Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 5′ untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA–protein interactions in both mitochondria and chloroplasts.

A Pentatricopeptide Repeat Protein Facilitates the trans-Splicing of the Maize Chloroplast rps12 Pre-mRNA

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.

The Pentatricopeptide Repeat Protein PPR5 Stabilizes a Specific tRNA Precursor in Maize Chloroplasts

ABSTRACT Genes for pentatricopeptide repeat (PPR) proteins are found in all eukaryotic genomes analyzed but are particularly abundant in land plants. The majority of analyzed PPR proteins play a role in the processing or translation of organellar RNAs. Few PPR proteins have been studied in detail, and the functional repertoire and mechanisms of action of proteins in the PPR family are poorly understood. Here we analyzed a maize ortholog of the embryo-essential Arabidopsis thaliana gene AtPPR5. A genome-wide analysis of chloroplast RNAs that coimmunoprecipitate with Zea mays PPR5 (ZmPPR5) demonstrated that ZmPPR5 is bound in vivo to the unspliced precursor of trnG-UCC. Null and hypomorphic Zmppr5 insertion mutants are embryo viable but are deficient for chloroplast ribosomes and die as seedlings. These mutants show a dramatic decrease in both spliced and unspliced trnG-UCC RNAs, while the transcription of trnG-UCC is unaffected. These results, together with biochemical data documenting the sequence-specific binding of recombinant PPR5 to the trnG-UCC group II intron, suggest that PPR5 stabilizes the trnG-UCC precursor by directly binding and protecting an endonuclease-sensitive site. These findings add to the evidence that chloroplast-localized PPR proteins that are embryo essential in Arabidopsis typically function in the biogenesis of the plastid translation machinery.

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain

Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self‐splicing activity was compensated by the recruitment of host‐encoded protein cofactors. Genetic screens previously identified CRS1 and CRS2 as host‐encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host‐encoded group II intron splicing factors, CRS2‐associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2–CAF1 and CRS2–CAF2 complexes have distinct intron specificities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNA‐binding module that has diversified to mediate specific interactions with various highly structured RNAs.

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.

Sequence-specific binding of a chloroplast pentatricopeptide repeat protein to its native group II intron ligand.

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 alpha 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 approximately 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.

A Ribonuclease III Domain Protein Functions in Group II Intron Splicing in Maize Chloroplasts

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.

Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize

High-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, bar-coded 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.

Effects of reduced chloroplast gene copy number on chloroplast gene expression in maize

Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Young chloroplasts contain hundreds of genome copies, but the functional significance of this high genome copy number has been unclear. We describe molecular phenotypes associated with mutations in a nuclear gene in maize (Zea mays), white2 (w2), encoding a predicted organellar DNA polymerase. Weak and strong mutant alleles cause a moderate (approximately 5-fold) and severe (approximately 100-fold) decrease in plastid DNA copy number, respectively, as assayed by quantitative PCR and Southern-blot hybridization of leaf DNA. Both alleles condition a decrease in most chloroplast RNAs, with the magnitude of the RNA deficiencies roughly paralleling that of the DNA deficiency. However, some RNAs are more sensitive to a decrease in genome copy number than others. The rpoB messenger RNA (mRNA) exhibited a unique response, accumulating to dramatically elevated levels in response to a moderate reduction in plastid DNA. Subunits of photosynthetic enzyme complexes were reduced more severely than were plastid mRNAs, possibly because of impaired translation resulting from limiting ribosomal RNA, transfer RNA, and ribosomal protein mRNA. These results indicate that chloroplast genome copy number is a limiting factor for the expression of a subset of chloroplast genes in maize. Whereas in Arabidopsis (Arabidopsis thaliana) a pair of orthologous genes function redundantly to catalyze DNA replication in both mitochondria and chloroplasts, the w2 gene is responsible for virtually all chloroplast DNA replication in maize. Mitochondrial DNA copy number was reduced approximately 2-fold in mutants harboring strong w2 alleles, suggesting that w2 also contributes to mitochondrial DNA replication.

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzymes properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.