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A protein-RNA specificity code enables targeted activation of an endogenous human transcript.

Campbell ZT, Valley CT, Wickens M - Nat. Struct. Mol. Biol. (2014)

Bottom Line: PUF proteins are an attractive platform for that purpose because they bind specific single-stranded RNA sequences by using short repeated modules, each contributing three amino acids that contact an RNA base.The resulting specificity code reveals the RNA binding preferences of natural proteins and enables the design of new specificities.Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.

ABSTRACT
Programmable protein scaffolds that target DNA are invaluable tools for genome engineering and designer control of transcription. RNA manipulation provides broad new opportunities for control, including changes in translation. PUF proteins are an attractive platform for that purpose because they bind specific single-stranded RNA sequences by using short repeated modules, each contributing three amino acids that contact an RNA base. Here, we identified the specificities of natural and designed combinations of those three amino acids, using a large randomized RNA library. The resulting specificity code reveals the RNA binding preferences of natural proteins and enables the design of new specificities. Using the code and a translational activation domain, we designed a protein that targets endogenous cyclin B1 mRNA in human cells, increasing sensitivity to chemotherapeutic drugs. Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.

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RNA recognition by the PUF proteins(A) The structure of C. elegans FBF-2 bound to RNA 9. RNA recognition is modular; each PUF repeat contributes an RNA recognition helix. (Inset) Three amino acid residues (referred to as a Tripartite Recognition Motif or TRM) form edge-on contacts (red lines) and stacking interactions (blue lines) with RNA bases (not all atoms are shown). By convention, the two edge-on residues are given in the same order in which they are found in the primary sequence (SE), followed by a dash and the stacking residue (H). (B) Abundance of natural TRMs inferred from sequence alignment. Pie charts represent TRM enrichment as a function of PUF repeat (R8-R1) based on 94 proteins.
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Figure 1: RNA recognition by the PUF proteins(A) The structure of C. elegans FBF-2 bound to RNA 9. RNA recognition is modular; each PUF repeat contributes an RNA recognition helix. (Inset) Three amino acid residues (referred to as a Tripartite Recognition Motif or TRM) form edge-on contacts (red lines) and stacking interactions (blue lines) with RNA bases (not all atoms are shown). By convention, the two edge-on residues are given in the same order in which they are found in the primary sequence (SE), followed by a dash and the stacking residue (H). (B) Abundance of natural TRMs inferred from sequence alignment. Pie charts represent TRM enrichment as a function of PUF repeat (R8-R1) based on 94 proteins.

Mentions: Comprehensive analysis of specificity among modular DNA binding proteins, including TAL effectors and zinc finger transcription factors, has led to powerful tools for genome engineering and manipulation of transcription 1,2. PUF (named for Pumilio and fem-3 binding factor) proteins provide an attractive scaffold with which to target RNAs instead, and enable access to new events, including the processing, transport, localization, translation and decay of messenger and non-coding RNAs. PUF proteins regulate mRNA expression through physical association with short (7-10 nucleotide) sequence elements 3-8. A single RNA base is discriminated by a bundle of three α-helices, typically present in eight tandem “PUF repeats” arranged in a semi-crescent (Fig. 1A) 9-12. The identity of the targeted base is dictated by the combination of three amino acid residues, referred to here as a tripartite recognition motif or TRM (Fig 1A). TRMs contact RNA bases through a combination of edge-on and stacking interactions 13.


A protein-RNA specificity code enables targeted activation of an endogenous human transcript.

Campbell ZT, Valley CT, Wickens M - Nat. Struct. Mol. Biol. (2014)

RNA recognition by the PUF proteins(A) The structure of C. elegans FBF-2 bound to RNA 9. RNA recognition is modular; each PUF repeat contributes an RNA recognition helix. (Inset) Three amino acid residues (referred to as a Tripartite Recognition Motif or TRM) form edge-on contacts (red lines) and stacking interactions (blue lines) with RNA bases (not all atoms are shown). By convention, the two edge-on residues are given in the same order in which they are found in the primary sequence (SE), followed by a dash and the stacking residue (H). (B) Abundance of natural TRMs inferred from sequence alignment. Pie charts represent TRM enrichment as a function of PUF repeat (R8-R1) based on 94 proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125476&req=5

Figure 1: RNA recognition by the PUF proteins(A) The structure of C. elegans FBF-2 bound to RNA 9. RNA recognition is modular; each PUF repeat contributes an RNA recognition helix. (Inset) Three amino acid residues (referred to as a Tripartite Recognition Motif or TRM) form edge-on contacts (red lines) and stacking interactions (blue lines) with RNA bases (not all atoms are shown). By convention, the two edge-on residues are given in the same order in which they are found in the primary sequence (SE), followed by a dash and the stacking residue (H). (B) Abundance of natural TRMs inferred from sequence alignment. Pie charts represent TRM enrichment as a function of PUF repeat (R8-R1) based on 94 proteins.
Mentions: Comprehensive analysis of specificity among modular DNA binding proteins, including TAL effectors and zinc finger transcription factors, has led to powerful tools for genome engineering and manipulation of transcription 1,2. PUF (named for Pumilio and fem-3 binding factor) proteins provide an attractive scaffold with which to target RNAs instead, and enable access to new events, including the processing, transport, localization, translation and decay of messenger and non-coding RNAs. PUF proteins regulate mRNA expression through physical association with short (7-10 nucleotide) sequence elements 3-8. A single RNA base is discriminated by a bundle of three α-helices, typically present in eight tandem “PUF repeats” arranged in a semi-crescent (Fig. 1A) 9-12. The identity of the targeted base is dictated by the combination of three amino acid residues, referred to here as a tripartite recognition motif or TRM (Fig 1A). TRMs contact RNA bases through a combination of edge-on and stacking interactions 13.

Bottom Line: PUF proteins are an attractive platform for that purpose because they bind specific single-stranded RNA sequences by using short repeated modules, each contributing three amino acids that contact an RNA base.The resulting specificity code reveals the RNA binding preferences of natural proteins and enables the design of new specificities.Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.

ABSTRACT
Programmable protein scaffolds that target DNA are invaluable tools for genome engineering and designer control of transcription. RNA manipulation provides broad new opportunities for control, including changes in translation. PUF proteins are an attractive platform for that purpose because they bind specific single-stranded RNA sequences by using short repeated modules, each contributing three amino acids that contact an RNA base. Here, we identified the specificities of natural and designed combinations of those three amino acids, using a large randomized RNA library. The resulting specificity code reveals the RNA binding preferences of natural proteins and enables the design of new specificities. Using the code and a translational activation domain, we designed a protein that targets endogenous cyclin B1 mRNA in human cells, increasing sensitivity to chemotherapeutic drugs. Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.

Show MeSH