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Phylogenetic and ontogenetic integration of organelles into the compartmentalized genome of the eukaryotic cell
Abstract and Figures
The chloroplast and mitochondrion genomes encode proteins important for organellar processes like photosynthesis, respiratory chain activity, and gene expression. Expression of organellar genes is regulated by numerous nuclear-encoded proteins involved in many posttranscriptional steps such as RNA processing, editing, splicing, stabilization, and translation. Many of these proteins are sequence specific RNA binding factors which are thought to recruit enzymes and lead them to the RNA target site rather than having enzymatic activity themselves. Here, we summarize the data of our recent research on global and specific nulear-encoded factors involved in organellar RNA meabolism in a general context.
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... Examples are PALE CRESS, ACCUMULATION OF PHOTOSYSTEM ONE1 (APO1) to APO4, PEPTIDE CHAIN RELEASE FACTOR B3 (PrfB3), RHO-N domain protein (RHON1), PLANT ORGANELLE RNA RECOGNITION domains, and many chloroplast PENTATRICOPEPTIDEREPEAT (PPR) proteins (Meurer et al., 1998;Kroeger et al., 2009;Stoppel et al., 2011Watkins et al., 2011;Shikanai and Fujii, 2013;Barkan and Small, 2014). Recruitment of novel factors for the management and regulation of the plastid and mitochondrial RNA metabolism was presumably driven by the acquisition of introns, massive endonucleolytic cleavage of precursor transcripts, numerous editing sites, and the fast divergence of noncoding sequences in the intergenic as well as the 59 and 39 UTRs in the genome of the endosymbiont (Barkan, 2011;Manavski et al., 2012a). Homologs of only few factors for chloroplast RNA metabolism are also found in cyanobacteria. ...
... Plastome-genome coevolution is an ongoing process that diverges in different lineages and retains species-specific interactions on the level of the RNA metabolism since it represents a fast-evolving process (Manavski et al., 2012a). Little is known about factors that authentically regulate plastid mRNA stability and even less about how metabolic processes as well as endogenous and external stimuli are involved in this regulation. ...
The seedling-lethal Arabidopsis thaliana high chlorophyll fluorescence145 (hcf145) mutation leads to reduced stability of the plastid tricistronic psaA-psaB-rps14 mRNA and photosystem I (PSI) deficiency. Here, we genetically mapped the HCF145 gene, which encodes a plant-specific, chloroplast-localized, modular protein containing two homologous domains related to the polyketide cyclase family comprising 37 annotated Arabidopsis proteins of unknown function. Two further highly conserved and previously uncharacterized tandem repeat motifs at the C terminus, herein designated the transcript binding motif repeat (TMR) domains, confer sequence-specific RNA binding capability to HCF145. Homologous TMR motifs are often found as multiple repeats in quite diverse proteins of green and red algae and in the cyanobacterium Microcoleus sp PCC 7113 with unknown function. HCF145 represents the only TMR protein found in vascular plants. Detailed analysis of hcf145 mutants in Arabidopsis and Physcomitrella patens as well as in vivo and in vitro RNA binding assays indicate that HCF145 has been recruited in embryophyta for the stabilization of the psaA-psaB-rps14 mRNA via specific binding to its 5' untranslated region. The polyketide cyclase-related motifs support association of the TMRs to the psaA RNA, presumably pointing to a regulatory role in adjusting PSI levels according to the requirements of the plant cell.
© 2015 American Society of Plant Biologists. All rights reserved.
... RNA metabolism represents a fast-evolving process. This progression depends mostly on vascular plant-specific, nuclear-encoded RNA-binding proteins and diverse conserved ribonucleases with their associated proteins (Jacobs and Kück, 2011;Manavski et al., 2012;Meurer, 2012, 2013). ...
The chloroplast hosts photosynthesis and a variety of metabolic pathways that are essential for plant viability and acclimation processes. In this study, we show that the sole plastid UMP kinase (PUMPKIN) in Arabidopsis (Arabidopsis thaliana) associates specifically with the introns of the plastid transcripts trnG-UCC, trnV-UAC, petB, petD, and ndhA in vivo, as revealed by RNA immunoprecipitation coupled with deep sequencing (RIP-Seq); and that PUMPKIN can bind RNA efficiently in vitro. Analyses of target transcripts showed that PUMPKIN affects their metabolism. Null alleles and knockdowns of pumpkin were viable but clearly affected in growth, plastid translation, and photosynthetic performance. In pumpkin mutants, the levels of many plastid transcripts were reduced, while the amounts of others were increased, as revealed by RNA-Seq analysis. PUMPKIN is a homomultimeric, plastid-localized protein that forms in vivo RNA-containing megadalton-sized complexes and catalyzes the ATP-dependent conversion of UMP to UDP in vitro with properties characteristic of known essential eubacterial UMP kinases. A moonlighting function of PUMPKIN combining RNA and pyrimidine metabolism is discussed. © 2019 American Society of Plant Biologists. All Rights Reserved.
... Most protectors are plant specific congruent with the fast divergence of their targets at the 5 -and 3 -UTRs of plastid transcripts [101]. Even if counterparts of the RNA-binding proteins are occasionally found in algae or even rarely in cyanobacteria, they exert a different function on transcripts or have different targets. ...
In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants.
The formation of chloroplasts can be traced back to an ancient event in which a eukaryotic host cell containing mitochondria ingested a cyanobacterium. Since then, chloroplasts have retained many characteristics of their bacterial ancestor, including their transcription and translation machinery. In this review, recent research on the maturation of rRNA and ribosome assembly in chloroplasts is explored, along with their crucial role in plant survival and their implications for plant acclimation to changing environments. A comparison is made between the ribosome composition and auxiliary factors of ancient and modern chloroplasts, providing insights into the evolution of ribosome assembly factors. Although the chloroplast contains ancient proteins with conserved functions in ribosome assembly, newly evolved factors have also emerged to help plants acclimate to changes in their environment and internal signals. Overall, this review offers a comprehensive analysis of the molecular mechanisms underlying chloroplast ribosome assembly and highlights the importance of this process in plant survival, acclimation, and adaptation.
Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5' untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5' ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5' maturation and translation initiation of rps3 mRNA.
The Arabidopsis endonuclease RNase E (RNE) is localized in the chloroplast and is involved in processing of plastid ribonucleic acids (RNAs).
By expression of a tandem affinity purification-tagged version of the plastid RNE in the Arabidopsis rne mutant background in combination with mass spectrometry, we identified the novel vascular plant-specific and co-regulated
interaction partner of RNE, designated RHON1. RHON1 is essential for photoautotrophic growth and together with RNE forms a
distinct ∼800 kDa complex. Additionally, RHON1 is part of various smaller RNA-containing complexes. RIP-chip and other association
studies revealed that a helix-extended-helix-structured Rho-N motif at the C-terminus of RHON1 binds to and supports processing
of specific plastid RNAs. In all respects, such as plastid RNA precursor accumulation, protein pattern, increased number and
decreased size of chloroplasts and defective chloroplast development, the phenotype of rhon1 knockout mutants resembles that of rne lines. This strongly suggests that RHON1 supports RNE functions presumably by conferring sequence specificity to the endonuclease.
The pentatricopeptide repeat (PPR) protein family is highly expanded in terrestrial plants. Arabidopsis contains 450 PPR genes, which represents 2% of the total protein-coding genes. PPR proteins are eukaryote-specific RNA-binding proteins implicated in multiple aspects of RNA metabolism of organellar genes. Most PPR proteins affect a single or small subset of gene(s), acting in a gene-specific manner. Studies over the last 10 years have revealed the significance of this protein family in coordinated gene expression in different compartments: the nucleus, chloroplast and mitochondrion. Here, we summarize recent studies addressing the mechanistic aspect of PPR proteins.
Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing
events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was
proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and
impedes 5′- and 3′-exonucleases, resulting in processed RNAs with PPR10 bound at the 5′- or 3′-end. In this study, we provide
evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map
additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein
HCF152 binds to the immediate 5′- or 3′-termini of transcripts that require HCF152 for their accumulation, providing evidence
that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding
sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA
termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint
of a PPR-like protein that protects the adjacent RNA from degradation.
Chloroplast biogenesis requires constant adjustment of RNA homeostasis under conditions of on-going developmental and environmental change and its regulation is achieved mainly by post-transcriptional control mechanisms mediated by various nucleus-encoded ribonucleases. More than 180 ribonucleases are annotated in Arabidopsis, but only 17 are predicted to localize to the chloroplast. Although different ribonucleases act at different RNA target sites in vivo, most nucleases that attack RNA are thought to lack intrinsic cleavage specificity and show non-specific activity in vitro. In vivo, specificity is thought to be imposed by auxiliary RNA-binding proteins, including members of the huge pentatricopeptide repeat family, which protect RNAs from non-specific nucleolytic attack by masking otherwise vulnerable sites. RNA stability is also influenced by secondary structure, polyadenylation, and ribosome binding. Ribonucleases may cleave at internal sites (endonucleases) or digest successively from the 5' or 3' end of the polynucleotide chain (exonucleases). In bacteria, RNases act in the maturation of rRNA and tRNA precursors, as well as in initiating the degradation of mRNAs and small non-coding RNAs. Many ribonucleases in the chloroplasts of higher plants possess homologies to their bacterial counterparts, but their precise functions have rarely been described. However, many ribonucleases present in the chloroplast process polycistronic rRNAs, tRNAs, and mRNAs. The resulting production of monocistronic, translationally competent mRNAs may represent an adaptation to the eukaryotic cellular environment. This review provides a basic overview of the current knowledge of RNases in plastids and highlights gaps to stimulate future studies.
Chloroplast RNA metabolism is controlled and excecuted by hundreds of nuclear-encoded, chloroplast-localized RNA binding proteins.
Contrary to the nucleo-cytosolic compartment or bacteria, there is little evidence for non-coding RNAs that play a role as
riboregulators of chloroplasts. We mined deep-sequencing datasets to identify short (16–28 nt) RNAs in the chloroplast genome
and found 50 abundant small RNAs (sRNAs) represented by multiple, in some cases, thousands of sequencing reads, whereas reads
are in general absent from the surrounding sequence space. Other than sRNAs representing the most highly abundant mRNAs, tRNAs
and rRNAs, most sRNAs are located in non-coding regions and many are found a short distance upstream of start codons. By transcript
end mapping we show that the 5′ and 3′ termini of chloroplast RNAs coincide with the ends of sRNAs. Sequences of sRNAs identified
in Arabidopsis are conserved between different angiosperm species and in several cases, we identified putative orthologs in rice deep sequencing
datasets. Recently, it was suggested that small chloroplast RNA fragments could result from the protective action of pentatricopeptide
repeat (PPR) proteins against exonucleases, i.e. footprints of RNA binding proteins. Our data support this scenario on a transcriptome-wide
level and suggest that a large number of sRNAs are in fact remnants of PPR protein targets.
Land plant genomes encode four functional ribosomal peptide chain release factors (Prf) of eubacterial origin, two (PrfA and PrfB homologs) for each endosymbiotic organelle. Formerly, we have shown that the Arabidopsis thaliana chloroplast-localized PrfB homolog, PrfB1, is required not only for termination of translation but also for stabilization of UGA stop codon-containing chloroplast transcripts. A previously undiscovered PrfB-like protein, PrfB3, is localized to the chloroplast stroma in a petB RNA-containing complex and found only in vascular plants. Highly conserved positions of introns unequivocally indicate that PrfB3 arose from a duplication of PrfB1. Notably, PrfB3 is lacking the two most important tripeptide motifs characteristic for all eubacterial and organellar PrfB homologs described so far: the stop codon recognition motif SPF and the catalytic center GGQ for peptidyl-tRNA hydrolysis. Complementation studies, as well as functional and molecular analyses of two allelic mutations in Arabidopsis, both of which lead to a specific deficiency of the cytochrome b₆f complex, revealed that PrfB3 is essentially required for photoautotrophic growth. Plastid transcript, polysome, and translation analyses indicate that PrfB3 has been recruited in vascular plants for light- and stress-dependent regulation of stability of 3' processed petB transcripts to adjust cytochrome b₆ levels.
Arabidopsis thaliana APO1 is required for the accumulation of the chloroplast photosystem I and NADH dehydrogenase complexes and had been proposed to facilitate the incorporation of [4Fe-4S] clusters into these complexes. The identification of maize (Zea mays) APO1 in coimmunoprecipitates with a protein involved in chloroplast RNA splicing prompted us to investigate a role for APO1 in splicing. We show here that APO1 promotes the splicing of several chloroplast group II introns: in Arabidopsis apo1 mutants, ycf3-intron 2 remains completely unspliced, petD intron splicing is strongly reduced, and the splicing of several other introns is compromised. These splicing defects can account for the loss of photosynthetic complexes in apo1 mutants. Recombinant APO1 from both maize and Arabidopsis binds RNA with high affinity in vitro, demonstrating that DUF794, the domain of unknown function that makes up almost the entirety of APO1, is an RNA binding domain. We provide evidence that DUF794 harbors two motifs that resemble zinc fingers, that these bind zinc, and that they are essential for APO1 function. DUF794 is found in a plant-specific protein family whose members are all predicted to localize to mitochondria or chloroplasts. Thus, DUF794 adds a new example to the repertoire of plant-specific RNA binding domains that emerged as a product of nuclear-organellar coevolution.
The conversion of genetic information stored in DNA into a protein product proceeds through the obligatory intermediate of messenger RNA. The steady-state level of an mRNA is determined by its relative synthesis and degradation rates, i.e., an interplay between transcriptional regulation and control of RNA stability. When the biological status of an organism requires that a gene product’s abundance varies as a function of developmental stage, environmental factors or intracellular signals, increased or decreased RNA stability can be the determining factor. RNA stability and processing have long been known as important regulatory points in chloroplast gene expression. Here we summarize current knowledge and prospects relevant to these processes, emphasizing biochemical data. The extensive literature on nuclear mutations affecting chloroplast RNA metabolism is reviewed in another article in this volume (Barkan and Goldschmidt-Clermont, this issue).