title: A protein-RNA specificity code enables targeted activation of an endogenous human transcript.
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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Campbell
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Cary T
authors.initials: CT
authors.lastname: Valley
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Marvin
authors.initials: M
authors.lastname: Wickens
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D056744:Cyclin B1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D014176:Protein Biosynthesis; D015202:Protein Engineering; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: C534699:CCNB1 protein, human; D056744:Cyclin B1; D012333:RNA, Messenger; D016601:RNA-Binding Proteins