Structure and RNA binding of the mouse Pumilio-2 Puf domain

ABSTRACTProgrammable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60-80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.

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Expanding the binding specificity for RNA recognition by a PUF domain

Nature Communications ◽

10.1038/s41467-021-25433-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wei Zhou ◽

Daniel Melamed ◽

Gabor Banyai ◽

Cindy Meyer ◽

Thomas Tuschl ◽

...

Keyword(s):

Rna Binding ◽

Amino Acid Substitutions ◽

Rna Recognition ◽

Specific Interactions ◽

Wild Type ◽

Modular Architecture ◽

Recognition Sequence ◽

Puf Domain ◽

Target Rna ◽

Type Sequence

AbstractThe ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

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Bespoke RNA recognition by Pumilios

Biochemical Society Transactions ◽

10.1042/bst20150072 ◽

2015 ◽

Vol 43 (5) ◽

pp. 801-806 ◽

Cited By ~ 4

Author(s):

Thomas A. Edwards

Keyword(s):

Rna Binding ◽

Consensus Sequence ◽

Rna Binding Protein ◽

Rna Recognition ◽

Binding Mechanism ◽

Recognition Sequence ◽

Single Base ◽

Puf Domain ◽

Binding Sequence

Pumilio is an RNA-binding protein originally identified in Drosophila, with a Puf domain made up of eight Puf repeats, three helix bundles arranged in a rainbow architecture, where each repeat recognizes a single base of the RNA-binding sequence. The eight-base recognition sequence can therefore be modified simply via mutation of the repeat that recognizes the base to be changed and this is understood in detail via high-resolution crystal structures. The binding mechanism is also altered in a variety of homologues from different species, with bases flipped out from the binding site to regenerate a consensus sequence. Thus Pumilios can be designed with bespoke RNA recognition sequences and can be fused to nucleases, split GFP, etc. as tools in vitro and in cells.

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