One C-to-U RNA Editing Site and Two Independently Evolved Editing Factors: Testing Reciprocal Complementation with DYW-Type PPR Proteins from the Moss Physcomitrium (Physcomitrella) patens and the Flowering Plants Macadamia integrifolia and Arabidopsis

Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as specificity factors through electrostatic interactions between a pair of polar residues and the nitrogenous bases of an RNA target. DYW-deaminase domains act as the editing enzyme. Two moss (Physcomitrella patens) PPR proteins containing DYW-deaminase domains, PPR65 and PPR56, can convert Cs to Us in cognate, exogenous RNA targets co-expressed in Escherichia coli. We show here that purified, recombinant PPR65 exhibits robust editase activity on synthetic RNAs containing cognate, mitochondrial PpccmFC sequences in vitro, indicating that a PPR protein with a DYW domain is solely sufficient for catalyzing C-to-U RNA editing in vitro. Monomeric fractions possessed the highest conversion efficiency, and oligomeric fractions had reduced activity. Inductively coupled plasma (ICP)–MS analysis indicated a stoichiometry of two zinc ions per highly active PPR65 monomer. Editing activity was sensitive to addition of zinc acetate or the zinc chelators 1,10-o-phenanthroline and EDTA. Addition of ATP or nonhydrolyzable nucleotide analogs stimulated PPR65-catalyzed RNA-editing activity on PpccmFC substrates, indicating potential allosteric regulation of PPR65 by ATP. Unlike for bacterial cytidine deaminase, addition of two putative transition-state analogs, zebularine and tetrahydrouridine, failed to disrupt RNA-editing activity. RNA oligonucleotides with a single incorporated zebularine also did not disrupt editing in vitro, suggesting that PPR65 cannot bind modified bases due to differences in the structure of the active site compared with other zinc-dependent nucleotide deaminases.

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Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606685113 ◽

2016 ◽

Vol 113 (43) ◽

pp. 12322-12327 ◽

Cited By ~ 74

Author(s):

Caterina Gerotto ◽

Alessandro Alboresi ◽

Andrea Meneghesso ◽

Martina Jokel ◽

Marjaana Suorsa ◽

...

Keyword(s):

Electron Transport ◽

Photosystem I ◽

Physcomitrella Patens ◽

Safety Valve ◽

Cell Metabolism ◽

Photosynthetic Apparatus ◽

Photosynthetic Electron Transport ◽

Flowering Plants ◽

Knock Out ◽

Electron Sink

Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens. We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV’s biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV’s role was taken by other alternative electron routes.

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Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6726-6734.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6726-6734 ◽

Cited By ~ 71

Author(s):

Tetsuya Miyamoto ◽

Junichi Obokata ◽

Masahiro Sugiura

Keyword(s):

Rna Editing ◽

Editing Site ◽

Mrna Editing ◽

Higher Plant ◽

In Vitro System ◽

Cis Acting ◽

Biochemical Identification ◽

Editing Activity

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

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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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