The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

Metabolic Labeling of RNAs Uncovers Hidden Features and Dynamics of the Arabidopsis Transcriptome.

Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants. Here, we demonstrate that RNA labeling with the modified, nontoxic uridine analog 5-ethynyl uridine (5-EU) in Arabidopsis (Arabidopsis thaliana) seedlings provides insight into plant transcriptome dynamics. Pulse labeling with 5-EU revealed nascent and unstable RNAs, RNA processing intermediates generated by splicing, and chloroplast RNAs. Pulse-chase experiments with 5-EU allowed us to determine RNA stabilities without the need for chemical transcription inhibitors such as actinomycin and cordycepin. Inhibitor-free, genome-wide analysis of polyadenylated RNA stability via 5-EU pulse-chase experiments revealed RNAs with shorter half-lives than those reported after chemical inhibition of transcription. In summary, our results indicate that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates and suggest that polyadenylated RNAs have low stability in plants. Our technique lays the foundation for easy, affordable, nascent transcriptome analysis and inhibitor-free analysis of RNA stability in plants.

Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

MAIN CONCLUSION: AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

KEY MESSAGE: Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

CRP1 Protein: (dis)similarities between Arabidopsis thaliana and Zea mays.

Biogenesis of chloroplasts in higher plants is initiated from proplastids, and involves a series of processes by which a plastid able to perform photosynthesis, to synthesize amino acids, lipids, and phytohormones is formed. All plastid protein complexes are composed of subunits encoded by the nucleus and chloroplast genomes, which require a coordinated gene expression to produce the correct concentrations of organellar proteins and to maintain organelle function. To achieve this, hundreds of nucleus-encoded factors are imported into the chloroplast to control plastid gene expression. Among these factors, members of the Pentatricopeptide Repeat (PPR) containing protein family have emerged as key regulators of the organellar post-transcriptional processing. PPR proteins represent a large family in plants, and the extent to which PPR functions are conserved between dicots and monocots deserves evaluation, in light of differences in photosynthetic metabolism (C3 vs. C4) and localization of chloroplast biogenesis (mesophyll vs. bundle sheath cells). In this work we investigated the role played in the process of chloroplast biogenesis by At5g42310, a member of the Arabidopsis PPR family which we here refer to as AtCRP1 (Chloroplast RNA Processing 1), providing a comparison with the orthologous ZmCRP1 protein from Zea mays. Loss-of-function atcrp1 mutants are characterized by yellow-albinotic cotyledons and leaves owing to defects in the accumulation of subunits of the thylakoid protein complexes. As in the case of ZmCRP1, AtCRP1 associates with the 5' UTRs of both psaC and, albeit very weakly, petA transcripts, indicating that the role of CRP1 as regulator of chloroplast protein synthesis has been conserved between maize and Arabidopsis. AtCRP1 also interacts with the petB-petD intergenic region and is required for the generation of petB and petD monocistronic RNAs. A similar role has been also attributed to ZmCRP1, although the direct interaction of ZmCRP1 with the petB-petD intergenic region has never been reported, which could indicate that AtCRP1 and ZmCRP1 differ, in part, in their plastid RNA targets.