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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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Modifications of PUF scaffolds using the TRM codeTRM variants are denoted as colored circles and RNAs as colors in bar charts according to the key. RNA sequences are provided for variant sites. Red nucleotides indicate sites that differ from the wild-type sequence. Binding activity measurements were conducted in the yeast-three hybrid system. (A) Replacement of TRMs in the PUM2 scaffold yields protein mutants with novel specificity. Most specificity mutants possessed binding activities comparable to that of the wild-type protein and RNA binding element. Error bars, s.d. (n = 3 independent colonies). (B) Mutations in FBF-2 yield altered specificity. Error bars, s.d. (n = 3 independent colonies). (C) Additivity of specificity mutations in PUM2. Sites of comparison between TRMs are highlighted in blue. Error bars, s.d. (n = 3 independent colonies).
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Figure 4: Modifications of PUF scaffolds using the TRM codeTRM variants are denoted as colored circles and RNAs as colors in bar charts according to the key. RNA sequences are provided for variant sites. Red nucleotides indicate sites that differ from the wild-type sequence. Binding activity measurements were conducted in the yeast-three hybrid system. (A) Replacement of TRMs in the PUM2 scaffold yields protein mutants with novel specificity. Most specificity mutants possessed binding activities comparable to that of the wild-type protein and RNA binding element. Error bars, s.d. (n = 3 independent colonies). (B) Mutations in FBF-2 yield altered specificity. Error bars, s.d. (n = 3 independent colonies). (C) Additivity of specificity mutations in PUM2. Sites of comparison between TRMs are highlighted in blue. Error bars, s.d. (n = 3 independent colonies).

Mentions: To determine how broadly the TRM code applies, we examined mutations in different repeats and scaffolds (Fig 4). We prepared ten RNA variants that contained one to six mutations in the RNA sequence that binds human PUM2. We engineered protein variants designed to bind these RNAs in a PUM2 scaffold, which had not been used to derive the TRM code. Wild-type PUM2 protein did not bind detectably to any of the mutant RNAs in yeast three-hybrid assays, though it did bind its wild-type site (Supplemental Fig 6). The engineered PUM2 proteins, designed using the TRM code, were tested against the same set of RNAs. We first introduced the best U-specific TRM (NQ–H) into repeat seven (Fig. 4A). This mutant protein bound the U-containing site and not the wild-type sequence. We then designed a series of additional substitutions using the most specific TRM for guanine recognition (SE–H), maintaining the U-specific Repeat 7. The resulting nine mutant proteins bound their novel cognate elements and not the wild-type sequence. This result was observed with as many as six mutations in the RNA elements. Similarly, in the FBF-2 scaffold, we tested binding of two double TRM-mutant proteins and one single TRM-mutant protein, each designed using the TRM code (Fig. 4B). These bound their targeted RNAs and not the wild-type site (Fig. 4B). We conclude that the TRM data are applicable to different scaffolds and repeats, and that they enable tailored recognition to three of the four RNA bases.


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

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

Modifications of PUF scaffolds using the TRM codeTRM variants are denoted as colored circles and RNAs as colors in bar charts according to the key. RNA sequences are provided for variant sites. Red nucleotides indicate sites that differ from the wild-type sequence. Binding activity measurements were conducted in the yeast-three hybrid system. (A) Replacement of TRMs in the PUM2 scaffold yields protein mutants with novel specificity. Most specificity mutants possessed binding activities comparable to that of the wild-type protein and RNA binding element. Error bars, s.d. (n = 3 independent colonies). (B) Mutations in FBF-2 yield altered specificity. Error bars, s.d. (n = 3 independent colonies). (C) Additivity of specificity mutations in PUM2. Sites of comparison between TRMs are highlighted in blue. Error bars, s.d. (n = 3 independent colonies).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125476&req=5

Figure 4: Modifications of PUF scaffolds using the TRM codeTRM variants are denoted as colored circles and RNAs as colors in bar charts according to the key. RNA sequences are provided for variant sites. Red nucleotides indicate sites that differ from the wild-type sequence. Binding activity measurements were conducted in the yeast-three hybrid system. (A) Replacement of TRMs in the PUM2 scaffold yields protein mutants with novel specificity. Most specificity mutants possessed binding activities comparable to that of the wild-type protein and RNA binding element. Error bars, s.d. (n = 3 independent colonies). (B) Mutations in FBF-2 yield altered specificity. Error bars, s.d. (n = 3 independent colonies). (C) Additivity of specificity mutations in PUM2. Sites of comparison between TRMs are highlighted in blue. Error bars, s.d. (n = 3 independent colonies).
Mentions: To determine how broadly the TRM code applies, we examined mutations in different repeats and scaffolds (Fig 4). We prepared ten RNA variants that contained one to six mutations in the RNA sequence that binds human PUM2. We engineered protein variants designed to bind these RNAs in a PUM2 scaffold, which had not been used to derive the TRM code. Wild-type PUM2 protein did not bind detectably to any of the mutant RNAs in yeast three-hybrid assays, though it did bind its wild-type site (Supplemental Fig 6). The engineered PUM2 proteins, designed using the TRM code, were tested against the same set of RNAs. We first introduced the best U-specific TRM (NQ–H) into repeat seven (Fig. 4A). This mutant protein bound the U-containing site and not the wild-type sequence. We then designed a series of additional substitutions using the most specific TRM for guanine recognition (SE–H), maintaining the U-specific Repeat 7. The resulting nine mutant proteins bound their novel cognate elements and not the wild-type sequence. This result was observed with as many as six mutations in the RNA elements. Similarly, in the FBF-2 scaffold, we tested binding of two double TRM-mutant proteins and one single TRM-mutant protein, each designed using the TRM code (Fig. 4B). These bound their targeted RNAs and not the wild-type site (Fig. 4B). We conclude that the TRM data are applicable to different scaffolds and repeats, and that they enable tailored recognition to three of the four RNA bases.

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
Related in: MedlinePlus