scholarly journals PPR-SMR protein SOT1 has RNA endonuclease activity

2017 ◽  
Vol 114 (8) ◽  
pp. E1554-E1563 ◽  
Author(s):  
Wen Zhou ◽  
Qingtao Lu ◽  
Qingwei Li ◽  
Lei Wang ◽  
Shunhua Ding ◽  
...  
Keyword(s):  
Endonuclease Activity ◽  
Pentatricopeptide Repeat ◽  
Rna Sequence ◽  
Ppr Protein ◽  
Specific Manner ◽  
Rna Endonuclease

Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), anArabidopsispentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5′ end of the chloroplast 23S–4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.

scholarly journals A PPR Protein ACM1 Is Involved in Chloroplast Gene Expression and Early Plastid Development in Arabidopsis

2021 ◽  
Vol 22 (5) ◽  
pp. 2512
Author(s):  
Xinwei Wang ◽  
Yaqi An ◽  
Ye Li ◽  
Jianwei Xiao
Keyword(s):  
Fluorescent Protein ◽  
Chloroplast Development ◽  
Nuclear Gene ◽  
Pentatricopeptide Repeat ◽  
Plastid Development ◽  
Chloroplast Gene Expression ◽  
Knock Out ◽  
Rnai Line ◽  
Ppr Protein ◽  
P Type

Chloroplasts cannot develop normally without the coordinated action of various proteins and signaling connections between the nucleus and the chloroplast genome. Many questions regarding these processes remain unanswered. Here, we report a novel P-type pentatricopeptide repeat (PPR) factor, named Albino Cotyledon Mutant1 (ACM1), which is encoded by a nuclear gene and involved in chloroplast development. Knock-down of ACM1 transgenic plants displayed albino cotyledons but normal true leaves, while knock-out of the ACM1 gene in seedlings was lethal. Fluorescent protein analysis showed that ACM1 was specifically localized within chloroplasts. PEP-dependent plastid transcript levels and splicing efficiency of several group II introns were seriously affected in cotyledons in the RNAi line. Furthermore, denaturing gel electrophoresis and Western blot experiments showed that the accumulation of chloroplast ribosomes was probably damaged. Collectively, our results indicate ACM1 is indispensable in early chloroplast development in Arabidopsis cotyledons.

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

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.

scholarly journals Comprehensive Transcriptome Analyses Reveal Candidate Genes for Variation in Seed Size/Weight During Peanut (Arachis hypogaea L.) Domestication

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhongfeng Li ◽  
Xingguo Zhang ◽  
Kunkun Zhao ◽  
Kai Zhao ◽  
Chengxin Qu ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Seed Development ◽  
Seed Size ◽  
Snp Markers ◽  
Pentatricopeptide Repeat ◽  
Significant Differential Expression ◽  
Ppr Protein ◽  
Cultivated Peanut ◽  
Transcriptome Analyses ◽  
Wild Peanut

Seed size/weight, a key domestication trait, is also an important selection target during peanut breeding. However, the mechanisms that regulate peanut seed development are unknown. We re-sequenced 12 RNA samples from developing seeds of two cultivated peanut accessions (Lines 8106 and 8107) and wild Arachis monticola at 15, 30, 45, and 60 days past flowering (DPF). Transcriptome analyses showed that ∼36,000 gene loci were expressed in each of the 12 RNA samples, with nearly half exhibiting moderate (2 ≤ FPKM < 10) expression levels. Of these genes, 12.2% (4,523) were specifically expressed during seed development, mainly at 15 DPF. Also, ∼12,000 genes showed significant differential expression at 30, 45, and/or 60 DPF within each of the three peanut accessions, accounting for 31.8–34.1% of the total expressed genes. Using a method that combined comprehensive transcriptome analysis and previously mapped QTLs, we identified several candidate genes that encode transcription factor TGA7, topless-related protein 2, IAA-amino acid hydrolase ILR1-like 5, and putative pentatricopeptide repeat-containing (PPR) protein. Based on sequence variations identified in these genes, SNP markers were developed and used to genotype both 30 peanut landraces and a genetic segregated population, implying that EVM0025654 encoding a PPR protein may be associated with the increased seed size/weight of the cultivated accessions in comparison with the allotetraploid wild peanut. Our results provide additional knowledge for the identification and functional research into candidate genes responsible for the seed size/weight phenotype in peanut.

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

2020 ◽  
Vol 71 (18) ◽  
pp. 5495-5505 ◽  
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.

scholarly journals 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

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.

scholarly journals Maize pentatricopeptide repeat protein DEK53 is required for mitochondrial RNA editing at multiple sites and seed development

2020 ◽  
Vol 71 (20) ◽  
pp. 6246-6261 ◽  
Author(s):  
Dawei Dai ◽  
Lifang Jin ◽  
Zhenzhen Huo ◽  
Shumei Yan ◽  
Zeyang Ma ◽  
...  
Keyword(s):  
Rna Editing ◽  
Protein Complexes ◽  
Plant Mitochondria ◽  
Mitochondrial Protein ◽  
Electron Microscopic Examination ◽  
Complex Iii ◽  
Mitochondrial Rna ◽  
Pentatricopeptide Repeat ◽  
Electron Microscopic ◽  
Ppr Protein

Abstract Pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing in plant mitochondria and plastids. In this study, we characterized maize (Zea mays) kernel mutant defective kernel 53 (dek53), which has an embryo lethal and collapsed endosperm phenotype. Dek53 encodes an E-subgroup PPR protein, which possesses a short PLS repeat region of only seven repeats. Subcellular localization analysis indicated that DEK53 is localized in the mitochondrion. Strand- and transcript-specific RNA-seq analysis showed that the dek53 mutation affected C-to-U RNA editing at more than 60 mitochondrial C targets. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the assembly of mitochondrial complex III in dek53. Transmission electron microscopic examination showed severe morphological defects of mitochondria in dek53 endosperm cells. In addition, yeast two-hybrid and luciferase complementation imaging assays indicated that DEK53 can interact with the mitochondrion-targeted non-PPR RNA editing factor ZmMORF1, suggesting that DEK53 might be a functional component of the organellar RNA editosome.

scholarly journals GUN1 and Plastid RNA Metabolism: Learning from Genetics

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2307
Author(s):  
Luca Tadini ◽  
Nicolaj Jeran ◽  
Paolo Pesaresi
Keyword(s):  
Rna Metabolism ◽  
Primary Role ◽  
Pentatricopeptide Repeat ◽  
Ppr Protein ◽  
Coordinated Expression ◽  
Unified View ◽  
Plastid Rna Polymerase ◽  
Flow Of Information ◽  
Opinion Article

GUN1 (genomes uncoupled 1), a chloroplast-localized pentatricopeptide repeat (PPR) protein with a C-terminal small mutS-related (SMR) domain, plays a central role in the retrograde communication of chloroplasts with the nucleus. This flow of information is required for the coordinated expression of plastid and nuclear genes, and it is essential for the correct development and functioning of chloroplasts. Multiple genetic and biochemical findings indicate that GUN1 is important for protein homeostasis in the chloroplast; however, a clear and unified view of GUN1′s role in the chloroplast is still missing. Recently, GUN1 has been reported to modulate the activity of the nucleus-encoded plastid RNA polymerase (NEP) and modulate editing of plastid RNAs upon activation of retrograde communication, revealing a major role of GUN1 in plastid RNA metabolism. In this opinion article, we discuss the recently identified links between plastid RNA metabolism and retrograde signaling by providing a new and extended concept of GUN1 activity, which integrates the multitude of functional genetic interactions reported over the last decade with its primary role in plastid transcription and transcript editing.

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

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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