The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript

Aryamanesh, Nader; Ruwe, Hannes; Sanglard, Lilian Vincis Pereira; Eshraghi, Leila; Bussell, John D.; Howell, Katharine A.; Small, Ian; Francs‐Small, Catherine Colas des

doi:10.1104/pp.16.01840

Cited by 51 publications

(82 citation statements)

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“…RNA footprinting has been used successfully to identify the binding sites of several PPR proteins, notably EMB2654, which binds to a sequence near the 3′ end of rps12 intron 1a (Aryamanesh et al ., ). We isolated short RNAs (sRNAs, potential footprints) from atppr4 plants, sequenced them and mapped the sequences to the plastid genome, in comparison with similar data from emb2654 mutants (Figure ).…”

Section: Resultsmentioning

confidence: 97%

“…As shown in Figure (e,h), there are subtle but reproducible differences in the sRNA coverage patterns in emb2654 and ppr4 mutants. The intron 1a footprint is more evident in ppr4 , which is consistent with the location of the binding site for EMB2654 within this region (Aryamanesh et al ., ). In contrast, the intron 1b footprint is more evident in emb2654 .…”

Section: Resultsmentioning

confidence: 97%

“…RUG3 (RCC1/UVR/GEF‐like 3) is important for the trans ‐splicing of nad2 intron 2 (Kühn et al ., ). In chloroplasts, the PPR proteins ZmPPR4 (Schmitz‐Linneweber et al ., ) and EMB2654 (Aryamanesh et al ., ), the CRM proteins CAF2 and CRS2 (Asakura and Barkan, ), and the RNA helicase (RH3) (Asakura et al ., ; Gu et al ., ) have been shown to be involved in the trans ‐splicing of rps12 intron 1. Here, we investigate the specific roles of PPR4 and EMB2654 in this process, which is crucial for chloroplast biogenesis and function, and ultimately for plant growth and development, and the extent to which these roles are likely to be conserved across land plants.…”

Section: Introductionmentioning

confidence: 99%

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The coordinated action of PPR4 and EMB2654 on each intron half mediates trans‐splicing of rps12 transcripts in plant chloroplasts

et al. 2019

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The pentatricopeptide repeat proteins PPR4 and EMB2654 have been shown to be required for the transsplicing of plastid rps12 transcripts in Zea mays (maize) and Arabidopsis, respectively, but their roles in this process are not well understood. We investigated the functions of the Arabidopsis and Oryza sativa (rice) orthologs of PPR4, designated AtPPR4 (At5g04810) and OsPPR4 (Os4g58780). Arabidopsis atppr4 and rice osppr4 mutants are embryo-lethal and seedling-lethal 3 weeks after germination, respectively, showing that PPR4 is essential in the development of both dicot and monocot plants. Artificial microRNA-mediated mutants of AtPPR4 displayed a specific defect in rps12 trans-splicing, with pale-green, yellowish or albino phenotypes, according to the degree of knock-down of AtPPR4 expression. Comparison of RNA footprints in atppr4 and emb2654 mutants showed a similar concordant loss of extensive footprints at the 3 0 end of intron 1a and at the 5 0 end of intron 1b in both cases. EMB2654 is known to bind within the footprint region in intron 1a and we show that AtPPR4 binds to the footprint region in intron 1b, via its PPR motifs. Binding of both PPR4 and EMB2654 is essential to juxtapose the two intron halves and to maintain the RNAs in a splicing-competent structure for the efficient trans-splicing of rps12 intron 1, which is crucial for chloroplast biogenesis and plant development. The similarity of EMB2654 and PPR4 orthologs and their respective binding sites across land plant phylogeny indicates that their coordinate function in rps12 trans-splicing has probably been conserved for 500 million years.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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The coordinated action of PPR4 and EMB2654 on each intron half mediates trans‐splicing of rps12 transcripts in plant chloroplasts

et al. 2019

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“…Although the phenotypes are different in details and highly variable depending on the type of mutated gene (there are many possibilities, e.g. genes for ribosomal proteins, tRNAs, rRNAs, translation factors, RNA processing factors and others), on the severity of the translation deficiency, on the phase of chloroplast development, when the translation deficiency starts to become effective, all mutants with impaired chloroplast translation show pigment deficiencies, lower performance of photosynthesis and altered thylakoid organization, often combined with retarded growth and delayed greening (Albrecht et al, 2006; Delannoy et al, 2009; Tiller and Bock, 2014; Liu et al, 2015; Kohler et al, 2016; Aryamanesh et al, 2017; Zhang et al, 2017). Another reason for proposing the ribosome deficiency as primary effect is that pigment deficiency, altered thylakoid organization or impaired photosynthesis does not cause chloroplast ribosome deficiencies, while the opposite occurs and can be explained by the function of chloroplast translation.…”

Section: Discussionmentioning

confidence: 99%

Mutation of theALBOSTRIANSOhnologous GeneHvCMF3Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

Hensel²,

Melzer³

et al. 2019

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Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained only if at least one gene of the pair undergoes neo- or subfunctionalization. Sequence-based phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed that they belong to a newly identified subfamily of genes encoding CCT domain proteins with putative N-terminal chloroplast transit peptides. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a xantha phenotype and successively develop pale green leaves. Compared to the wild type, plastids of the mutant seedlings show decreased PSII efficiency and lower amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional region, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs localized to plastids. Hvcmf3Hvcmf7 double mutants indicated epistatic activity of HvCMF7 over HvCMF3. The chloroplast ribosome deficiency is discussed as the primary defect of the Hvcmf3 mutants. Our data suggests that HvCMF3 and HvCMF7 have similar but not identical functions.One-sentence summaryPhylogenetic and mutant analyses of the barley protein HvCMF3 (ALBOSTRIANS-LIKE) identified, in higher plants, a subfamily of CCT domain proteins with essential function in chloroplast development.

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“…In contrast, defects in Rps8 translation due to loss of PGR3 in not4a likely explains why ribosome depletion is specific to the 30S subunit, since protein complex stoichiometries are highly regulated, with the inability to assemble complete complexes often leading to degradation of orphan subunits (Juszkiewicz and Hegde, 2018; Taggart et al, 2020). In addition, upregulation of PPR proteins SOT1, EMB2654, PPR4 and PPR2 (which all promote chloroplast rRNA maturation), and GUN1 (implicated in the production of chloroplast ribosomal subunits RPS1 and RPL11) present further evidence of compensatory responses to reduced ribosome abundance and protein synthesis within not4a chloroplasts (Figure S6B)(Aryamanesh et al, 2017; Lee et al, 2019; Lu et al, 2011; Tadini et al, 2016; Wu et al, 2016).…”

Section: Resultsmentioning

confidence: 97%

TheArabidopsisNOT4A E3 ligase coordinates PGR3 expression to regulate chloroplast protein translation

Bailey

Ivanauskaite

Grimmer

et al. 2020

Preprint

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31Chloroplast function requires the coordinated action of nuclear-and chloroplast-derived 32 proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) 33 proteins that regulate plastid mRNA metabolism. Despite their large number and importance, 34 regulatory mechanisms controlling PPR expression are poorly understood. Here we show 35 that the Arabidopsis NOT4A ubiquitin-ligase positively regulates PROTON GRADIENT 3 36 (PGR3), a PPR protein required for translating 30S ribosome subunits and several thylakoid-37 localised photosynthetic components within chloroplasts. Loss of NOT4A function leads to a 38 strong depletion of plastid ribosomes, which reduces mRNA translation and negatively 39 impacts photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. 40Quantitative transcriptome and proteome analyses reveal that these defects are due to a 41 lack of PGR3 expression in not4a, and we show that normal plastid function is restored 42 through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring 43 robust photosynthetic function during development and stress-response, through modulating 44 PGR3 levels to coordinate chloroplast protein synthesis.The synthesis of energy from the sun, photosynthesis, supports organic life on earth. Light 68 harvesting in green plants takes place within the specialized chloroplast organelle, believed 69 to have arisen from engulfment of a photosynthetic prokaryote by an ancestral eukaryotic 70 cell (Archibald, 2015). Coevolution and merging of these organisms has resulted in nuclear 71 and chloroplast genomes separated within cellular compartments. In land plants, the 72 chloroplast genome comprises of ~130 genes, yet chloroplasts contain around 3000 different 73 proteins (Zoschke and Bock, 2018). Consequently, chloroplast function requires expression 74 not only of chloroplast encoded proteins, but a multitude of nuclear encoded genes, which 75 are imported into chloroplasts post-translationally. One such group of nuclear derived factors 76 is the pentatricopeptide repeat domain (PPR) containing proteins. The PPR protein family 77 has significantly expanded in plants (~450 in Arabidopsis, vs <10 in humans and yeast; 78 (Schmitz-Linneweber and Small, 2008)), and members are characterized by a 35-amino acid 79 repeat sequence that facilitates RNA binding and enables them to provide critical gene 80 expression control within chloroplasts and mitochondria (Barkan and Small, 2014). Through 81 binding to organellar RNAs, PPR proteins stabilize gene transcripts, facilitate post-82 transcriptional processing and promote translation of the encoded proteins (Barkan and 83 Small, 2014; Manna, 2015). Whilst their function in the regulation of gene expression control 84 within organelles has been described, including many of the RNA species to which they 85 bind, little is known about how their expression is regulated prior to import. 86Precise, selective removal of proteins is essential to cellular development and response. In 87...

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The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript

Cited by 51 publications

References 77 publications

The coordinated action of PPR4 and EMB2654 on each intron half mediates trans‐splicing of rps12 transcripts in plant chloroplasts

The coordinated action of PPR4 and EMB2654 on each intron half mediates trans‐splicing of rps12 transcripts in plant chloroplasts

Mutation of theALBOSTRIANSOhnologous GeneHvCMF3Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

TheArabidopsisNOT4A E3 ligase coordinates PGR3 expression to regulate chloroplast protein translation

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