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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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Engineered specificity of PUF proteins(A) Sequence motifs of wild-type (WT) and redesigned (neo-PUF) proteins. The targeted recognition site possesses a single substitution at position seven of the binding element. (B) Analysis of RNA binding activity in a yeast three-hybrid assay. RNA binding for wild-type (blue) and neo-PUF (orange) measurements for an empty vector, positive control gld-1 RNA element, and the cyclin B1 targeting element are shown. Error bars, s.d. (n = 3 independent colonies). (C) Binding enrichment values for the neo-PUF and the wild-type proteins were calculated for the in vitro consensus sequence HUGURWWWU and subtracted (Wt-Neo) 4. On the vertical axis are arrayed 384 sequences – a subset of the 410 possible 10-mers analyzed computationally – arranged in logical order by sequence. A subset of the sequences shown on the right, posses sequences altered at positions +7 and +9. The plots indicate the degree of enrichment either for the wild-type or neo-PUF proteins. Values are shaded as in A, with positive enrichments shaded green to red and negative enrichments shaded light to dark blue.
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Figure 5: Engineered specificity of PUF proteins(A) Sequence motifs of wild-type (WT) and redesigned (neo-PUF) proteins. The targeted recognition site possesses a single substitution at position seven of the binding element. (B) Analysis of RNA binding activity in a yeast three-hybrid assay. RNA binding for wild-type (blue) and neo-PUF (orange) measurements for an empty vector, positive control gld-1 RNA element, and the cyclin B1 targeting element are shown. Error bars, s.d. (n = 3 independent colonies). (C) Binding enrichment values for the neo-PUF and the wild-type proteins were calculated for the in vitro consensus sequence HUGURWWWU and subtracted (Wt-Neo) 4. On the vertical axis are arrayed 384 sequences – a subset of the 410 possible 10-mers analyzed computationally – arranged in logical order by sequence. A subset of the sequences shown on the right, posses sequences altered at positions +7 and +9. The plots indicate the degree of enrichment either for the wild-type or neo-PUF proteins. Values are shaded as in A, with positive enrichments shaded green to red and negative enrichments shaded light to dark blue.

Mentions: To explore the utility of TRM data for manipulation of mRNA expression, we engineered FBF-2 to bind a specific RNA sequence in the 3’UTR of human cyclin B1 mRNA. Cyclin B1 is a critical regulator of the cell cycle responsible for entry into mitosis and exit from G2 26,27. We altered repeat 3 of FBF-2 so that it should now bind a sequence in the 3'UTR of cyclin B1 mRNA, in which position 7 is non-consensus (UGUGUUUU). We refer to this protein as a “neo-PUF” (Fig. 5A). The in vitro consensus differs slightly from the consensus derived from yeast three-hybrid studies, UGUANNAU 18. Both SEQRS and yeast three-hybrid assays revealed that the neo-PUF now bound the desired element, with PUF repeat 3 binding U rather than A (Fig. 5A and 5B). To globally analyze differences in specificity between the wild-type and neo-PUF, we subtracted the enrichment value of sequences obtained with the neo-PUF from those obtained with the wild-type protein; thus negative values indicate preferential binding to the neo-PUF and positive values, preferential binding to the wild-type protein (Fig. 5C). Careful inspection of a subset of these sequences provides an example, using a region in which only the identities of position 7 and 9 vary. +7U sequences were enriched by the neo-PUF, while the wild-type protein enriched +7A, regardless of the larger sequence context. Enrichment oscillates along the axis, indicating that changes at position +7 (and in the highlighted case, not at +9) dictate the enrichment of a given sequence. We conclude that the TRM data are applicable to accurately predict modified specificity at alternate PUF repeats.


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

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

Engineered specificity of PUF proteins(A) Sequence motifs of wild-type (WT) and redesigned (neo-PUF) proteins. The targeted recognition site possesses a single substitution at position seven of the binding element. (B) Analysis of RNA binding activity in a yeast three-hybrid assay. RNA binding for wild-type (blue) and neo-PUF (orange) measurements for an empty vector, positive control gld-1 RNA element, and the cyclin B1 targeting element are shown. Error bars, s.d. (n = 3 independent colonies). (C) Binding enrichment values for the neo-PUF and the wild-type proteins were calculated for the in vitro consensus sequence HUGURWWWU and subtracted (Wt-Neo) 4. On the vertical axis are arrayed 384 sequences – a subset of the 410 possible 10-mers analyzed computationally – arranged in logical order by sequence. A subset of the sequences shown on the right, posses sequences altered at positions +7 and +9. The plots indicate the degree of enrichment either for the wild-type or neo-PUF proteins. Values are shaded as in A, with positive enrichments shaded green to red and negative enrichments shaded light to dark blue.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Engineered specificity of PUF proteins(A) Sequence motifs of wild-type (WT) and redesigned (neo-PUF) proteins. The targeted recognition site possesses a single substitution at position seven of the binding element. (B) Analysis of RNA binding activity in a yeast three-hybrid assay. RNA binding for wild-type (blue) and neo-PUF (orange) measurements for an empty vector, positive control gld-1 RNA element, and the cyclin B1 targeting element are shown. Error bars, s.d. (n = 3 independent colonies). (C) Binding enrichment values for the neo-PUF and the wild-type proteins were calculated for the in vitro consensus sequence HUGURWWWU and subtracted (Wt-Neo) 4. On the vertical axis are arrayed 384 sequences – a subset of the 410 possible 10-mers analyzed computationally – arranged in logical order by sequence. A subset of the sequences shown on the right, posses sequences altered at positions +7 and +9. The plots indicate the degree of enrichment either for the wild-type or neo-PUF proteins. Values are shaded as in A, with positive enrichments shaded green to red and negative enrichments shaded light to dark blue.
Mentions: To explore the utility of TRM data for manipulation of mRNA expression, we engineered FBF-2 to bind a specific RNA sequence in the 3’UTR of human cyclin B1 mRNA. Cyclin B1 is a critical regulator of the cell cycle responsible for entry into mitosis and exit from G2 26,27. We altered repeat 3 of FBF-2 so that it should now bind a sequence in the 3'UTR of cyclin B1 mRNA, in which position 7 is non-consensus (UGUGUUUU). We refer to this protein as a “neo-PUF” (Fig. 5A). The in vitro consensus differs slightly from the consensus derived from yeast three-hybrid studies, UGUANNAU 18. Both SEQRS and yeast three-hybrid assays revealed that the neo-PUF now bound the desired element, with PUF repeat 3 binding U rather than A (Fig. 5A and 5B). To globally analyze differences in specificity between the wild-type and neo-PUF, we subtracted the enrichment value of sequences obtained with the neo-PUF from those obtained with the wild-type protein; thus negative values indicate preferential binding to the neo-PUF and positive values, preferential binding to the wild-type protein (Fig. 5C). Careful inspection of a subset of these sequences provides an example, using a region in which only the identities of position 7 and 9 vary. +7U sequences were enriched by the neo-PUF, while the wild-type protein enriched +7A, regardless of the larger sequence context. Enrichment oscillates along the axis, indicating that changes at position +7 (and in the highlighted case, not at +9) dictate the enrichment of a given sequence. We conclude that the TRM data are applicable to accurately predict modified specificity at alternate PUF repeats.

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