Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells

Yagi, Yusuke; Teramoto, Takamasa; Kaieda, Shuji; Imai, Takayoshi; Sasaki, Tadamasa; Yagi, Masayuki; Maekawa, Nana; Nakamura, Takahiro

doi:10.3390/cells11223529

Cited by 5 publications

(9 citation statements)

References 47 publications

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“…Previous applications of synthetic PPR proteins include use as an affinity tag to immunopurify a specific chloroplast mRNP (McDermoi et al, 2019), substitution for endogenous PPR RNA stabilization factors in chloroplasts (Manavski et al, 2021), regulation of splice site choice in mammalian cells (Yagi et al, 2022), substitution for an endogenous chloroplast PPR RNA editing factor (Royan et al, 2021) and editing of that same chloroplast RNA sequence when expressed in E. coli (Bernath-Levin et al, 2021). Our findings add translational activation to the repertoire of functions that can be programmed with sPPR proteins.…”

Section: Discussionmentioning

confidence: 99%

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Translational activation by a synthetic PPR protein elucidates control ofpsbAtranslation in Arabidopsis chloroplasts

Rojas,

Chotewutmontri,

Barkan

2024

Preprint

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Translation initiation on psbA mRNA in plant chloroplasts scales with light intensity, providing its gene product, D1, to replace photodamaged D1 in Photosystem II. The psbA translational activator HCF173 has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with the psbA 5'-untranslated region (5'-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173's footprint in the psbA 5'-UTR bound the intended site in vivo and partially substituted for HCF173 to activate psbA translation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulate psbA translation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts.

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Section: Discussionmentioning

confidence: 99%

“…Recent advances in sPPR protein design, fostered by the development of high throughput screening methods (Bernath-Levin et al, 2021; Yagi et al, 2022), hold promise for overcoming these challenges.…”

Section: Discussionmentioning

confidence: 99%

Translational activation by a synthetic PPR protein elucidates control ofpsbAtranslation in Arabidopsis chloroplasts

Rojas,

Chotewutmontri,

Barkan

2024

Preprint

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“…For example, many natural PPR proteins are implicated in RNA splicing, but how they act in these processes is too uncertain for the time being to engineer PPR proteins to predictably influence plant organellar RNA splicing. However, PPR proteins can be used to control alternative splicing in mammalian cells by deliberately targeting the PPRs at sequences required for exon recognition ( Yagi et al. 2022 ).…”

Section: Synthetic Ppr Proteinsmentioning

confidence: 99%

“…In this study, the authors used synthetic PPR proteins to promote exon-skipping in transcripts encoding a bi-chromatic fluorescent reporter protein in HEK293T cells. They went on to demonstrate that the same approach could work to influence exon-skipping of endogenous mRNAs in the same cells ( Yagi et al. 2022 ).…”

Section: Synthetic Ppr Proteinsmentioning

confidence: 99%

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Applications of Synthetic Pentatricopeptide Repeat Proteins

Kwok van der Giezen,

Honkanen,

Colas des Francs-Small

et al. 2023

Plant and Cell Physiology

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RNA-binding proteins play integral roles in the regulation of essential processes in cells and as such are attractive targets for engineering to manipulate gene expression at the RNA level. Expression of transcripts in chloroplasts and mitochondria is heavily regulated by pentatricopeptide repeat (PPR) proteins. The diverse roles of PPR proteins and their naturally modular architecture make them ideal candidates for engineering. Synthetic PPR proteins are showing great potential to become valuable tools for controlling the expression of plastid and mitochondrial transcripts. In this review, by ‘synthetic’, we mean both rationally modified natural PPR proteins and completely novel proteins designed using the principles learned from their natural counterparts. We focus on the many different applications of synthetic PPR proteins, covering both their use in basic research to learn more about protein–RNA interactions and their use to achieve specific outcomes in RNA processing and the control of gene expression. We describe the challenges associated with the design, construction and deployment of synthetic PPR proteins and provide perspectives on how they might be assembled and used in future biotechnology applications.

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Translational enhancement of target endogenous mRNA in mammalian cells using programmable RNA-binding pentatricopeptide repeat proteins

Ping,

Hara-Kuge,

Yagi

et al. 2024

Sci Rep

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Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism.

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Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells

Cited by 5 publications

References 47 publications

Translational activation by a synthetic PPR protein elucidates control ofpsbAtranslation in Arabidopsis chloroplasts

Translational activation by a synthetic PPR protein elucidates control ofpsbAtranslation in Arabidopsis chloroplasts

Applications of Synthetic Pentatricopeptide Repeat Proteins

Translational enhancement of target endogenous mRNA in mammalian cells using programmable RNA-binding pentatricopeptide repeat proteins

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