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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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Manipulation of translation using a modified PUF scaffold(A) Effects of a neo-activator on Cyclin B1 levels in U2OS cells measured by western blotting. (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05, by two-tailed Student's t test. (B) Immunofluorescence of Cyclin B1 expression in U2OS cells expressing the Neo-activator (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test. Slides were observed under the same microscope using identical parameters (scale bar = 100 μm). (C) Viability assays. Viability was quantified 24 hours post-treatment. Normalized cell death is shown for two M-phase targeting drugs 27. Cyclin B1 was overexpressed as a positive control. Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test.
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Figure 6: Manipulation of translation using a modified PUF scaffold(A) Effects of a neo-activator on Cyclin B1 levels in U2OS cells measured by western blotting. (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05, by two-tailed Student's t test. (B) Immunofluorescence of Cyclin B1 expression in U2OS cells expressing the Neo-activator (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test. Slides were observed under the same microscope using identical parameters (scale bar = 100 μm). (C) Viability assays. Viability was quantified 24 hours post-treatment. Normalized cell death is shown for two M-phase targeting drugs 27. Cyclin B1 was overexpressed as a positive control. Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test.

Mentions: To enhance translation of endogenous cyclin B1, we fused a 20 kDa segment of yeast poly(A) binding protein (PAB) to the neo-PUF protein. This domain stimulates translation of a reporter in Xenopus laevis oocytes 29. We refer to this chimera as a “neo-activator.” The neo-activator increased Cyclin B1 protein abundance by approximately 400 percent; neither the RNA-binding defective form fused to PAB (termed RNADEF-PAB) nor vector alone did so (Supplemental Fig. 8). The levels of protein expression of the neo-activator and the RNA-binding defective form were comparable (Supplementary Fig. 7A). The neo-PUF without the PAB moiety had little effect on Cyclin B1 levels, demonstrating that the PUF scaffold was functionally inert (Supplemental Fig. 8). Increased Cyclin B1 protein abundance was confirmed by immunofluorescence spectroscopy, in which the fraction of Cyclin B1 positive cells elevated by approximately 500 percent (Fig. 6B).


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

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

Manipulation of translation using a modified PUF scaffold(A) Effects of a neo-activator on Cyclin B1 levels in U2OS cells measured by western blotting. (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05, by two-tailed Student's t test. (B) Immunofluorescence of Cyclin B1 expression in U2OS cells expressing the Neo-activator (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test. Slides were observed under the same microscope using identical parameters (scale bar = 100 μm). (C) Viability assays. Viability was quantified 24 hours post-treatment. Normalized cell death is shown for two M-phase targeting drugs 27. Cyclin B1 was overexpressed as a positive control. Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test.
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Figure 6: Manipulation of translation using a modified PUF scaffold(A) Effects of a neo-activator on Cyclin B1 levels in U2OS cells measured by western blotting. (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05, by two-tailed Student's t test. (B) Immunofluorescence of Cyclin B1 expression in U2OS cells expressing the Neo-activator (right - quantification). Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test. Slides were observed under the same microscope using identical parameters (scale bar = 100 μm). (C) Viability assays. Viability was quantified 24 hours post-treatment. Normalized cell death is shown for two M-phase targeting drugs 27. Cyclin B1 was overexpressed as a positive control. Error bars, s.d. (n = 3 cell cultures) * P < 0.05, ** P < 0.05 by two-tailed Student's t test.
Mentions: To enhance translation of endogenous cyclin B1, we fused a 20 kDa segment of yeast poly(A) binding protein (PAB) to the neo-PUF protein. This domain stimulates translation of a reporter in Xenopus laevis oocytes 29. We refer to this chimera as a “neo-activator.” The neo-activator increased Cyclin B1 protein abundance by approximately 400 percent; neither the RNA-binding defective form fused to PAB (termed RNADEF-PAB) nor vector alone did so (Supplemental Fig. 8). The levels of protein expression of the neo-activator and the RNA-binding defective form were comparable (Supplementary Fig. 7A). The neo-PUF without the PAB moiety had little effect on Cyclin B1 levels, demonstrating that the PUF scaffold was functionally inert (Supplemental Fig. 8). Increased Cyclin B1 protein abundance was confirmed by immunofluorescence spectroscopy, in which the fraction of Cyclin B1 positive cells elevated by approximately 500 percent (Fig. 6B).

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