Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids

Recent identification of several different types of RNA editing factors in plant organelles suggests complex RNA editosomes within which each factor has a different task. However, the precise protein interactions between the different editing factors are still poorly understood. In this paper, we show that the E+-type pentatricopeptide repeat (PPR) protein SLO2, which lacks a C-terminal cytidine deaminase-like DYW domain, interacts in vivo with the DYW-type PPR protein DYW2 and the P-type PPR protein NUWA in mitochondria, and that the latter enhances the interaction of the former ones. These results may reflect a protein scaffold or complex stabilization role of NUWA between E+-type PPR and DYW2 proteins. Interestingly, DYW2 and NUWA also interact in chloroplasts, and DYW2-GFP overexpressing lines show broad editing defects in both organelles, with predominant specificity for sites edited by E+-type PPR proteins. The latter suggests a coordinated regulation of organellar multiple site editing through DYW2, which probably provides the deaminase activity to E+ editosomes.

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Maize pentatricopeptide repeat protein DEK41 affects cis-splicing of mitochondrial nad4 intron 3 and is required for normal seed development

Journal of Experimental Botany ◽

10.1093/jxb/erz193 ◽

2019 ◽

Vol 70 (15) ◽

pp. 3795-3808 ◽

Cited By ~ 14

Author(s):

Chenguang Zhu ◽

Guangpu Jin ◽

Peng Fang ◽

Yan Zhang ◽

Xuzhen Feng ◽

...

Keyword(s):

Protein Interactions ◽

Mitochondrial Rna ◽

Pentatricopeptide Repeat ◽

Lethal Mutant ◽

Developmental Defects ◽

Ppr Protein ◽

Normal Seed ◽

Ppr Proteins ◽

P Type ◽

Complex I Assembly

Abstract The splicing of organelle-encoded mRNA in plants requires proteins encoded in the nucleus. The mechanism of splicing and the factors involved are not well understood. Pentatricopeptide repeat (PPR) proteins are known to participate in such RNA–protein interactions. Maize defective kernel 41 (dek41) is a seedling-lethal mutant that causes developmental defects. In this study, the Dek41 gene was cloned by Mutator tag isolation and allelic confirmation, and was found to encode a P-type PPR protein that targets mitochondria. Analysis of the mitochondrial RNA transcript profile revealed that dek41 mutations cause reduced splicing efficiency of mitochondrial nad4 intron 3. Immature dek41 kernels exhibited severe reductions in complex I assembly and NADH dehydrogenase activity. Up-regulated expression of alternative oxidase genes and deformed inner cristae of mitochondria in dek41, as revealed by TEM, indicated that proper splicing of nad4 is essential for correct mitochondrial functioning and morphology. Consistent with this finding, differentially expressed genes in the dek41 endosperm included those related to mitochondrial function and activity. Our results indicate that DEK41 is a PPR protein that affects cis-splicing of mitochondrial nad4 intron 3 and is required for correct mitochondrial functioning and maize kernel development.

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In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

10.1101/2020.11.21.392746 ◽

2020 ◽

Author(s):

Nikolay Manavski ◽

Louis-Valentin Meteignier ◽

Margarita Rojas ◽

Andreas Brachmann ◽

Alice Barkan ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Specificity ◽

Pentatricopeptide Repeat ◽

Functional Capabilities ◽

Ppr Protein ◽

Repeat Proteins ◽

Ppr Proteins ◽

Physical Barriers

ABSTRACTPentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5’ end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5’ UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

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A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

10.21203/rs.3.rs-114803/v1 ◽

2020 ◽

Author(s):

Santana Royan ◽

Bernard Gutmann ◽

Catherine Colas des Francs-Small ◽

Suvi Honkanen ◽

Jason Schmidberger ◽

...

Keyword(s):

Rna Editing ◽

Protein Interactions ◽

Rna Binding ◽

Catalytic Domain ◽

Rna Binding Proteins ◽

Cytidine Deaminase ◽

Land Plant ◽

Target Sequence ◽

Pentatricopeptide Repeat

Abstract Targeted cytidine to uridine RNA editing is a widespread phenomenon throughout the land plant lineage. Members of the pentatricopeptide repeat (PPR) protein family act as the specificity factors in this process. These proteins consist of helix-turn-helix domains, each of which recognises a single RNA nucleotide following a well-elucidated code. A cytidine deaminase-like domain (present at the C-terminus of some PPR editing factors or provided in trans via protein-protein interactions) is the catalytic domain in the process. The huge expansion of the PPR superfamily in land plants provides the sequence variation required for design of novel consensus-based RNA-binding proteins. We used this approach to construct a synthetic RNA editing factor designed to target one of the two sites in the Arabidopsis chloroplast transcriptome naturally recognised by the RNA editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that this designed editing factor specifically recognises the target sequence in in vitro binding assays and can partially complement a clb19 mutant. The designed factor is specific for the target rpoA site and does not recognise or edit the other site recognised by CLB19 in the clpP1 transcript. We show that the designed editing factor can function equally specifically in the bacterium E. coli, and shows some activity even in the absence of the editing cofactors that are often required for natural editing factor activity in plants. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

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The DYW-subgroup pentatricopeptide repeat protein PPR27 interacts with ZmMORF1 to facilitate mitochondrial RNA editing and seed development in maize

Journal of Experimental Botany ◽

10.1093/jxb/eraa273 ◽

2020 ◽

Vol 71 (18) ◽

pp. 5495-5505 ◽

Cited By ~ 1

Author(s):

Rui Liu ◽

Shi-Kai Cao ◽

Aqib Sayyed ◽

Huan-Huan Yang ◽

Jiao Zhao ◽

...

Keyword(s):

Rna Editing ◽

Seed Development ◽

Plant Mitochondria ◽

Endosperm Development ◽

Complex Iii ◽

Mitochondrial Rna ◽

Mitochondrial Complex ◽

Pentatricopeptide Repeat ◽

Ppr Protein ◽

Ppr Proteins

Abstract C-to-U RNA editing in plant mitochondria requires the participation of many nucleus-encoded factors, most of which are pentatricopeptide repeat (PPR) proteins. There is a large number of PPR proteins and the functions many of them are unknown. Here, we report a mitochondrion-localized DYW-subgroup PPR protein, PPR27, which functions in the editing of multiple mitochondrial transcripts in maize. The ppr27 mutant is completely deficient in C-to-U editing at the ccmFN-1357 and rps3-707 sites, and editing at six other sites is substantially reduced. The lack of editing at ccmFN-1357 causes a deficiency of CcmFN protein. As CcmFN functions in the maturation pathway of cytochrome proteins that are subunits of mitochondrial complex III, its deficiency results in an absence of cytochrome c1 and cytochrome c proteins. Consequently, the assembly of mitochondrial complex III and super-complex I+III2 is decreased, which impairs the electron transport chain and respiration, leading to arrests in embryogenesis and endosperm development in ppr27. In addition, PPR27 was found to physically interact with ZmMORF1, which interacts with ZmMORF8, suggesting that these three proteins may facilitate C-to-U RNA editing via the formation of a complex in maize mitochondria. This RNA editing is essential for complex III assembly and seed development in maize.

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The novel E-subgroup pentatricopeptide repeat protein DEK55 is responsible for RNA editing at multiple sites and for the splicing of nad1 and nad4 in maize

BMC Plant Biology ◽

10.1186/s12870-020-02765-x ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Ru Chang Ren ◽

Xu Wei Yan ◽

Ya Jie Zhao ◽

Yi Ming Wei ◽

Xiaoduo Lu ◽

...

Keyword(s):

Rna Editing ◽

Rna Processing ◽

Mitochondrial Gene ◽

Endosperm Development ◽

Molecular Evidence ◽

Pentatricopeptide Repeat ◽

Reading Frame ◽

Maize Kernel ◽

Ppr Protein ◽

Ppr Proteins

Abstract Background Pentatricopeptide repeat (PPR) proteins compose a large protein family whose members are involved in both RNA processing in organelles and plant growth. Previous reports have shown that E-subgroup PPR proteins are involved in RNA editing. However, the additional functions and roles of the E-subgroup PPR proteins are unknown. Results In this study, we developed and identified a new maize kernel mutant with arrested embryo and endosperm development, i.e., defective kernel (dek) 55 (dek55). Genetic and molecular evidence suggested that the defective kernels resulted from a mononucleotide alteration (C to T) at + 449 bp within the open reading frame (ORF) of Zm00001d014471 (hereafter referred to as DEK55). DEK55 encodes an E-subgroup PPR protein within the mitochondria. Molecular analyses showed that the editing percentage of 24 RNA editing sites decreased and that of seven RNA editing sites increased in dek55 kernels, the sites of which were distributed across 14 mitochondrial gene transcripts. Moreover, the splicing efficiency of nad1 introns 1 and 4 and nad4 intron 1 significantly decreased in dek55 compared with the wild type (WT). These results indicate that DEK55 plays a crucial role in RNA editing at multiple sites as well as in the splicing of nad1 and nad4 introns. Mutation in the DEK55 gene led to the dysfunction of mitochondrial complex I. Moreover, yeast two-hybrid assays showed that DEK55 interacts with two multiple organellar RNA-editing factors (MORFs), i.e., ZmMORF1 (Zm00001d049043) and ZmMORF8 (Zm00001d048291). Conclusions Our results demonstrated that a mutation in the DEK55 gene affects the mitochondrial function essential for maize kernel development. Our results also provide novel insight into the molecular functions of E-subgroup PPR proteins involved in plant organellar RNA processing.

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Non-canonical Features of Pentatricopeptide Repeat Protein-Facilitated RNA Editing in Arabidopsis Chloroplasts

10.1101/544486 ◽

2019 ◽

Author(s):

Yueming Kelly Sun ◽

Bernard Gutmann ◽

Ian Small

Keyword(s):

Rna Editing ◽

Plant Mitochondria ◽

Pentatricopeptide Repeat ◽

Site Specific ◽

Ppr Protein ◽

Sequence Variations ◽

Ppr Proteins ◽

Editing Activity ◽

Chloroplast Rna ◽

Plant Organelles

AbstractCytosine (C) to uracil (U) RNA editing in plant mitochondria and chloroplasts is facilitated by site-specific pentatricopeptide repeat (PPR) editing factors. PPR editing factors contain multiple types of PPR motifs, and PPR motifs of the same type also show sequence variations. Therefore, no PPR motifs are invariant within a PPR protein or between different PPR proteins. This work evaluates the functional diversity of PPR motifs in CHLOROPLAST RNA EDITING FACTOR 3 (CREF3). The results indicate that previously overlooked features of PPR editing factors could also contribute to RNA editing activity. In particular, the N-terminal degenerated PPR motifs and the two L1-type PPR motifs in CREF3 are functionally indispensable. Furthermore, PPR motifs of the same type in CREF3 are not interchangeable. These non-canonical features of CREF3 have important implications on the understanding of PPR-facilitated RNA editing in plant organelles.

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Cofactor-Independent RNA Editing by a Synthetic S-Type PPR Protein

Synthetic Biology ◽

10.1093/synbio/ysab034 ◽

2021 ◽

Author(s):

Kalia Bernath-Levin ◽

Jason Schmidberger ◽

Suvi Honkanen ◽

Bernard Gutmann ◽

Yueming Kelly Sun ◽

...

Keyword(s):

Rna Editing ◽

Rna Processing ◽

Tandem Repeats ◽

Rna Binding ◽

Rna Binding Proteins ◽

Screening Method ◽

Pentatricopeptide Repeat ◽

Wide Range ◽

Ppr Proteins ◽

P Type

ABSTRACT Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors, and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method we show that synthetic S-type PPR proteins are easy to design, bind with high affinity and specificity, and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

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The Amount of RNA Editing Sites in Liverwort Organellar Genes Is Correlated with GC Content and Nuclear PPR Protein Diversity

Genome Biology and Evolution ◽

10.1093/gbe/evz232 ◽

2019 ◽

Vol 11 (11) ◽

pp. 3233-3239 ◽

Cited By ~ 3

Author(s):

Shanshan Dong ◽

Chaoxian Zhao ◽

Shouzhou Zhang ◽

Hong Wu ◽

Weixue Mu ◽

...

Keyword(s):

Rna Editing ◽

Phylogenetic Diversity ◽

Gc Content ◽

Pentatricopeptide Repeat ◽

Protein Coding ◽

Protein Diversity ◽

Ppr Protein ◽

Ppr Proteins ◽

Codon Positions ◽

Positive Correlations

Abstract RNA editing occurs in the organellar mRNAs of all land plants but the marchantioid liverworts, making liverworts a perfect group for studying the evolution of RNA editing. Here, we profiled the RNA editing of 42 exemplars spanning the ordinal phylogenetic diversity of liverworts, and screened for the nuclear-encoded pentatricopeptide repeat (PPR) proteins in the transcriptome assemblies of these taxa. We identified 7,428 RNA editing sites in 128 organellar genes from 31 non-marchantioid liverwort species, and characterized 25,059 PPR protein sequences. The abundance of organellar RNA editing sites varies greatly among liverwort lineages, genes, and codon positions, and shows strong positive correlations with the GC content of protein-coding genes, and the diversity of the PLS class of nuclear PPR proteins.

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A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

Communications Biology ◽

10.1038/s42003-021-02062-9 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Santana Royan ◽

Bernard Gutmann ◽

Catherine Colas des Francs-Small ◽

Suvi Honkanen ◽

Jason Schmidberger ◽

...

Keyword(s):

Rna Editing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Target Sequence ◽

Pentatricopeptide Repeat ◽

Binding Assays ◽

E Coli ◽

Ppr Protein ◽

Ppr Proteins

AbstractMembers of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

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GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean

Frontiers in Plant Science ◽

10.3389/fpls.2021.690973 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xingxing Feng ◽

Suxin Yang ◽

Yaohua Zhang ◽

Cheng Zhiyuan ◽

Kuanqiang Tang ◽

...

Keyword(s):

Rna Editing ◽

Pentatricopeptide Repeat ◽

Mobility Shift ◽

Single Nucleotide ◽

Chlorophyll Contents ◽

Complex Processes ◽

Ppr Protein ◽

Ppr Proteins ◽

Mobility Shift Assay

Chloroplast biogenesis and development are highly complex processes requiring interactions between plastids and nuclear genomic products. Pentatricopeptide repeat (PPR) proteins play an essential role in the development of chloroplasts; however, it remains unclear how RNA editing factors influence soybean development. In this study, a Glycine max pale green leaf 2 mutant (Gmpgl2) was identified with decreased chlorophyll contents. Genetic mapping revealed that a single-nucleotide deletion at position 1949 bp in the Glyma.05g132700 gene in the Gmpgl2 mutant, resulting in a truncated GmPGL2 protein. The nuclear-encoded GmPGL2 is a PLS-type PPR protein that localizes to the chloroplasts. The C-to-U editing efficiencies of rps16, rps18, ndhB, ndhD, ndhE, and ndhF were reduced in the Gmpgl2 mutant. RNA electrophoresis mobility shift assay (REMSA) analysis further revealed that GmPGL2 binds to the immediate upstream sequences at RNA editing sites of rps16 and ndhB in vitro, respectively. In addition, GmPGL2 was found to interact with GmMORF8, GmMORF9, and GmORRM6. These results suggest that GmPGL2 participates in C-to-U RNA editing via the formation of a complex RNA editosome in soybean chloroplasts.

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