Introduction
Plant organelles, including chloroplasts and mitochondria, derived from free-living cyanobacteria and α-proteobacteria (; ). During the evolution, the massive organellar genes were transferred to the nuclear genome, resulting in only 3-209 proteins are encoded from the current chloroplast or mitochondrial genome (). The organelles-encoded proteins are crucial for organellar gene expression (OGE), photosynthesis, organellar electron transport chain, cellular metabolism, and ATP production (). Notably, OGE is chiefly regulated at post-transcriptional steps, including RNA cleavage, RNA stability, RNA editing (C to U), and RNA splicing (; ). The fine-tuned regulation of organellar post-transcriptional RNA metabolism essentially require hundreds of the nucleus-encoded chloroplast and mitochondrial RNA-binding proteins (nCMRBPs), including pentatricopeptide repeat (PPR) proteins, chloroplast ribosome maturation and splicing domain (CRM) proteins, DEAD-Box RNA helicases (RHs), S1 domain-containing proteins (SDPs), and mitochondrial transcription termination factors (mTERFs), during the entire period of plant growth and development (; ; ; ). Many recent studies have demonstrated that the lack of these proteins leads to the defective RNA metabolsim in plants under various environmental stresses (; ).
PPR proteins are extensively distributed to plant lineages, containing more than 400 family members, and function as organellar-specific RNA-binding proteins (; ; ). Interestingly, a redesign of PPR tracts was shown to recognize programmed RNA targets and to control organellar gene expression (; Yagi et al., 2014; ; ). Here, we highlight a novel idea to redesign the PPR tracts of PPR4 and PPR19 proteins, which could affect the affinity of cognate RNAs in plant’s response to developmental and environmental cues.
Mechanism of P-type PPR proteins for target RNA recognition
The PPR constitutes a pair of antiparallel α helices of degenerate 35-amino acid tandem repeated-motifs ranging from 2 to 30 tracts (; ). The binding specificity of PPR motifs relies on the basis of 1 repeat to 1 nucleotide binding module, leading to the association of single-stranded RNA (; ). P-class PPR proteins mainly consist of pure PPR motifs, which are important for organellar transcript processing, RNA stabilization, and group II intron splicing (; ; Wang et al., 2021). The PPR-RNA binding codes show that the amino acid residues at the fifth and thirty-fifth positions are essential for sequence specificity in the association of PPR protein-RNA substrate (; ; ; Yan et al., 2019). Importantly, amino acid codes that recognize the specific RNA bases harbor side chains playing an important role for hydrogen bond interaction with the similar manner of Watson and Crick base paring (; ; ), suggesting further possibility to predict and/or customize target RNA sequences via the combinational PPR-RNA binding code.
Redesign of artificial PPR proteins in organelles
To date, the usage of amino acid codes for nucleotide recognition suggests that modifying amino acid of PPR motifs causes a weak or strong ability in perceiving target RNA substrates (; Yin et al., 2013; ; Yan et al., 2019). Indeed, artificial PPRs exhibit the ability of recognizing programmed RNA targets and can modulate chloroplast gene expression (; Yagi et al., 2014). For instance, ZmPPR10 recognizing 5’ UTR of chloroplast ZmatpH was modified to successfully switch on the nuclear transgene expression in tobacco plastids and potato amyloplasts (; Yu et al., 2019). The native PPR proteins of dPPRrbcL and dPPRpetL were tailored to be associated with the specific sites in the 5’ UTR of rbcL and petL mRNAs in Arabidopsis chloroplasts, respectively (). The RPF2, an Arabidopsis mitochondrial PPR protein, specifically binds to two cognate RNA sites located within the 5’ UTRs of cox3 and nad9 genes (). Interestingly, the reprogrammed PPR tracts of RFP2 protein, which were designed to bind new target RNA sequences within the open reading frame of nad6, recognized the new RNA target site and subsequently led to a cleavage of the RNA molecule in vivo (). These results demonstrate the specificities and in vivo functionalities of the artificial PPR proteins.
Engineering of PPR4 and PPR19 proteins for organellar gene regulation
Previous studies showed that PPR19, which includes tandem-repeated 19 PPR tracts, is crucial for the stabilization of nad1 intron 3a in Arabidopsis mitochondria and PPR4, which possesses one RNA-recognition motif (RRM) and tandem-repeated 15 PPR tracts, plays an essential role in the splicing of rps12 intron 1b in Arabidopsis chloroplasts (; ). Although the RRM and PPR motifs of PPR4 are associated with the strong RNA-binding activity of the PPR4 protein, the PPR motifs alone can strongly bind to the specific sequences of rps12 intron 1b (). Importantly, the designed 11 and 14 PPR tracts showed a similar binding affinity to intended target substrates (), whereas the longer repeated-tracts tend to promote off-target action and reduce target-binding specificity. Notably, previous researches focused on switching on transgenes in chloroplasts or targeting to new RNA substrates in chloroplasts and mitochondria by combinations of manipulated PPR motifs (; ; ; Yu et al., 2019; ). However, we suggest that rdPPR4 and rdPPR19 proteins are manipulated in its motifs for promoting the affinity of the proteins to its cognate RNAs (Figure 1). We anticipate that these optimizations will enhance the splicing of chloroplast rps12 intron 1 and the stability of nad1 intron 3 in native target location, which will increase the function of photosystems and mitochondrial electron transfer chains in plants under optimal conditions and environmental cues. The improvement of organellar functions will confer the phenotypic tolerance in plant’s response to abiotic stresses, including high salinity, drought, extreme temperatures, and high light.
Figure 1
Conclusion and perspectives
Engineering PPR proteins can be harnessed for the manipulation of intron splicing and/or stabilization of organellar RNA molecules, and these redesigned PPR proteins will be a potential means to improve plant’s fitness to developmental and environmental cues. Engineered PPR proteins can endow the PPR proteins with a scaffolder for RNA targets, which can be potentially applicable for synthetic and RNA biology. Engineering PPR proteins combined with the artificial organelles-trafficking system can be utilized in agricultural crops to produce or restore cytoplasmic male sterility (CMS) which maternally fails to produce fertile pollens due to abnormalities of mitochondrial RNA metabolism.
Statements
Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.
Funding
This research was supported by Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2019R1A6A1A10072987) to KL and by a grant from the Mid-career Researcher Program through the NRF of Korea funded by the Ministry of Science, ICT and Future Planning (NRF-2021R1A2C1004187) to HK, Republic of Korea.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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Summary
Keywords
engineering PPR proteins, organellar gene expression, organellar RNA metabolism, chloroplast, mitochondria
Citation
Lee K and Kang H (2023) Engineering of pentatricopeptide repeat proteins in organellar gene regulation. Front. Plant Sci. 14:1144298. doi: 10.3389/fpls.2023.1144298
Received
17 January 2023
Accepted
21 February 2023
Published
01 March 2023
Volume
14 - 2023
Edited by
Jirong Huang, Shanghai Institute for Biological Sciences (CAS), China
Reviewed by
Ping Yin, Huazhong Agricultural University, China
Updates
Copyright
© 2023 Lee and Kang.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Kwanuk Lee, kulee@jejunu.ac.kr; Hunseung Kang, hskang@jnu.ac.kr
This article was submitted to Plant Development and EvoDevo, a section of the journal Frontiers in Plant Science
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.