Programmable RNA-binding protein composed of repeats of a single modular unit

Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Boyden, Edward S.

doi:10.1073/pnas.1519368113

Cited by 58 publications

(55 citation statements)

References 51 publications

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“…Adamala et al. concatenated single PUF repeats in chain to target desired RNA sequences and showed that repeat 6 could be a template for the canonical modules . Therefore, repeat 6 might have high structural tolerance for the neighboring repeat, which leads to preferable binding activity of insertion‐type 16‐repeat PUFs.…”

Section: Resultsmentioning

confidence: 99%

Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation

et al. 2017

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RNA-binding proteins recognizing unique sequences within large transcriptomes serve as a powerful tool to control RNA metabolism. Pumilio and fem-3 mRNA-binding factor (PUF) proteins are considered good candidates for such tools, because they are typically composed of eight highly homologous repeat segments and can be designed to recognize arbitrary 8 nt RNA sequences. However, a specific 8 nt RNA sequence is found at multiple sites in various RNAs in the transcriptome, making it difficult to specifically target a single RNA. Designer PUF proteins recognizing longer RNA sequences should achieve more selective binding. Here, we propose an approach for creating 16-repeat PUFs capable of targeting a single, unique mRNA in the transcriptome. Our design is simple and involves either the tandem alignment of two PUF segments or the nesting of one PUF segment within another. Designed 16-repeat PUFs bound to the target RNA sequence without partial recognition derived from the original 8-repeat PUF. Furthermore, based on our strategy, expression of an endogenous mRNA was selectively and effectively modulated, demonstrating the applicability of 16-repeat PUF proteins for regulating endogenous RNA metabolism.

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Section: Resultsmentioning

confidence: 99%

Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation

et al. 2017

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“…The RNA specificity of the PUF domain has been well decoded (44)(45)(46). Based on the properties of the PUF proteins, the programmable RNA binding by the engineered PUF protein has been realized (47)(48)(49)(50). Moreover, the PUF domains fusing with a general RNA cleavage domain have been generated to the artificial site-specific RNA endonucleases that specifically recognize and cleave RNA targets (51,52).…”

Section: Discussionmentioning

confidence: 99%

PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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), an Arabidopsis pentatricopeptide 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.photosynthesis | PPR-SMR protein | RNA endonuclease | rRNA biogenesis S equence-specific RNA endonucleases are crucial to establishing RNA manipulation technology (1). Compared with DNA editing, RNA manipulation could be more useful and reversible because it does not result in permanent changes to the genome. In addition, sequence-specific RNA endonucleases could potentially be used as an RNA silencing tool to complement RNAi, because RNAi is sometimes ineffective in certain organisms and RNAi machinery is not present in cellular compartments such as chloroplasts and mitochondria. Despite extensive investigations, a natural RNA endonuclease that recognizes RNA in an intrinsic sequence-specific manner has not yet been identified.Pentatricopeptide repeat (PPR) proteins exist in eukaryotes, have greatly expanded in terrestrial plants, and take part in most RNA metabolic processes in organelles (2-5). The PPR domain can specifically recognize RNAs in an intrinsic sequence-specific manner (5-8). The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19). Recent studies demonstrated the SMR domain exhibits DNA nicking nuclease activity in vitro (20-25). Furthermore, a C-terminal SMR domain in Leishmania donovani S-phase mRNA cycling sequence binding protein (CSBP) has RNA cleavage activity in vitro (26). These findings suggest that the SMR domain has nuclease activity.Interestingly, a small protein family containing both PPR and SMR domains was recently described (27). PPR-SMR proteins are found mainly in land plants. Arabidopsis thaliana contains eight PPR-SMR proteins localized to the organelles, including mitochondria and chloroplasts (27). Currently, four PPR-SMR proteins have been characterized. They play ...

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“…So far, we have only demonstrated the activity of PAM2 motifs by tethered function analysis. However, PAM2 peptides also may be combined with sequence-specific RNA-binding molecules, such as Pumilio homology domains5051, CRISPR-Cas95253 or antisense oligonucleotides54 to suppress NMD independent of tethering. Applied in combination with read-through drugs, PAM2 peptides may also enable to restore the expression of nonsense-mutated genes.…”

Section: Discussionmentioning

confidence: 99%

Harnessing short poly(A)-binding protein-interacting peptides for the suppression of nonsense-mediated mRNA decay

Fatscher¹,

Gehring²

2016

Sci Rep

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Nonsense-mediated mRNA decay (NMD) is a cellular process that eliminates messenger RNA (mRNA) substrates with premature translation termination codons (PTCs). In addition, NMD regulates the expression of a number of physiological mRNAs, for example transcripts containing long 3′ UTRs. Current models implicate the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and translation termination in NMD. Accordingly, PABPC1 present within close proximity of a termination codon antagonizes NMD. Here, we use reporter mRNAs with different NMD-inducing 3′ UTRs to establish a general NMD-inhibiting property of PABPC1. NMD-inhibition is not limited to PABPC1, but can also be achieved by peptides consisting of the PABP-interacting motif 2 (PAM2) of different proteins when recruited to an NMD-inhibiting position of NMD reporter transcripts. The short PAM2 peptides efficiently suppress NMD activated by a long 3′ UTR, an exon-junction complex (EJC) and individual EJC components, and stabilize a PTC-containing β-globin mRNA. In conclusion, our results establish short PABPC1-recruiting peptides as potent but position-dependent inhibitors of mammalian NMD.

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Programmable RNA-binding protein composed of repeats of a single modular unit

Cited by 58 publications

References 51 publications

Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation

Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation

PPR-SMR protein SOT1 has RNA endonuclease activity

Harnessing short poly(A)-binding protein-interacting peptides for the suppression of nonsense-mediated mRNA decay

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