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

Royan, Santana; Gutmann, Bernard; Francs‐Small, Catherine Colas des; Honkanen, Suvi; Schmidberger, J.W.; Soet, Ashley; Sun, Yueming K.; Sanglard, Lilian Vincis Pereira; Bond, Charles S.; Small, Ian

doi:10.1038/s42003-021-02062-9

Cited by 38 publications

(42 citation statements)

References 59 publications

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“…A very recent new approach has focused on particular S-type PPRs ( 78 ) modeling the binding properties of the widely studied natural CLB19 editing factor ( 50 , 78–85 ). This work extended and complemented an earlier attempt using a synthetic PLS-type PPR array as a surrogate for the PPR array of CLB19, which, however, turned out to require MORF2, a ‘Multiple Organelle RNA editing Factor’, as a necessary co-factor for obtaining moderate RNA editing efficiency ( 86 ). Using the E. coli assay setup, the synthetic PLS-type PPR protein did not cause an off-target editing, while the artificial S-type approach resulted in 50% of RNA editing at its natural CLB19 rpoA target and only weakly affected one single off-target ( tufB ) in E. coli ( 78 ).…”

Section: Discussionmentioning

confidence: 56%

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

Lesch

Schilling

Brenner

et al. 2022

Nucleic Acids Research

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RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.

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Section: Discussionmentioning

confidence: 56%

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

Lesch

Schilling

Brenner

et al. 2022

Nucleic Acids Research

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“…MORF proteins are essential for effective RNA editing in angiosperms because they interact with PPR domains at the L motif and increase their affinity to target RNAs (Yan et al., 2017). RNA editing with synthetic PLS type PPR proteins in E. coli required co‐expression of MORF2 (Royan et al., 2021).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the design of synthetic PPR‐type RNA editing factors based on consensus motifs was reported (Royan et al., 2021). This approach is very attractive in terms of developing programmable biotechnological tools to convert specific cytidines to uridines in RNA.…”

Section: Discussionmentioning

confidence: 99%

“…We developed a parallel expression system for a PPR editing factor and its target(s) from two individual plasmids. A two‐plasmid expression system for E. coli RNA editing was reported using two pET‐based vectors for expressing two RNA editing factors, PPR and MORF, in an E. coli cell (Royan et al., 2021). Because the co‐existence of two plasmids containing the same replication origin is often unstable and it is difficult to control the copy numbers of each plasmid, our system using plasmids with distinct replication origins has an advantage in this respect.…”

Section: Discussionmentioning

confidence: 99%

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DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites

et al. 2022

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C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5 0 sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the À1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.

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“…In-silico data (Cheng et al, 2016;Harrison et al, 2016;Shen et al, 2019) indicate the preferred sequence 5'-UACCAAAAGGU for MSP1's PPR 4-11 motifs. (B) The PPRmatcher algorithm (Royan et al, 2021), which aligns the predicated PPR motifs to every possible position in the mitogenome of Arabidopsis (i.e., ~720K positions) and scores each alignment based on the data in (Yan et al, 2019).…”

Section: Data Availabilitymentioning

confidence: 99%

MSP1encodes an essential RNA-binding PPR factor required fornad1maturation and complex I biogenesis inArabidopsismitochondria

Best

Mizrahi

Edris

et al. 2022

Preprint

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Mitochondria are semi-autonomous organelles that serve as hubs for aerobic energy metabolism. The biogenesis of the respiratory (OXPHOS) system relies on nuclear-encoded factors, which regulate the transcription, processing and translation of mitochondrial (mt)RNAs. These include proteins of primordial origin, as well as eukaryotic-type RNA-binding families recruited from the host genomes to function in mitogenome expression. Pentatricopeptide repeat (PPR) proteins constitute a major gene-family in angiosperms that is pivotal in many aspects of mtRNA metabolism, such as editing, splicing or stability. Here, we report the analysis ofMITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1(MSP1, At4g20090), a canonical mitochondria-localized PPR protein that is necessary for mitochondrial biogenesis and embryo-development. Functional complementation confirmed that the phenotypes result from a disruption of theMSP1gene. As a loss-of-function allele ofArabidopsis MSP1leads to seed abortion, we employed an embryo-rescue method for the molecular characterization ofmsp1mutants. Our data show that msp1 embryo-development fails to proceed beyond the heart-torpedo transition stage as a consequence of a severenad1pre-RNA processing-defect, resulting in the loss of respiratory complex I (CI) activity. The maturation of nad1 involves the processing of three RNA-fragments,nad1.1,nad1.2andnad1.3. Based on detailed analyses of the mtRNA profiles in wild-type andmsp1plants, we concluded that through its association with a specific site innad1.1, MSP1 facilitates the generation of its 3'-terminus and stabilizes it - a prerequisite fornad1exons a-b splicing. Our data substantiate the importance of mtRNA metabolism for the biogenesis of the respiratory machinery during early-plant development.

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

Cited by 38 publications

References 59 publications

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites

MSP1encodes an essential RNA-binding PPR factor required fornad1maturation and complex I biogenesis inArabidopsismitochondria

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