title: An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.
abstract: Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA.
authors.forename: David W
authors.initials: DW
authors.lastname: Crawford
authors.forename: Brett D
authors.initials: BD
authors.lastname: Blakeley
authors.affiliation: Department of Surgery and Department of Biochemistry & Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine , Los Angeles, California 90033, United States.
authors.forename: Po-Han
authors.initials: PH
authors.lastname: Chen
authors.affiliation: Basic Research Laboratory, National Cancer Institute , Frederick, Maryland 21702, United States.
authors.forename: Chringma
authors.initials: C
authors.lastname: Sherpa
authors.affiliation: Basic Research Laboratory, National Cancer Institute , Frederick, Maryland 21702, United States.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.affiliation: Department of Surgery and Department of Biochemistry & Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine , Los Angeles, California 90033, United States.
authors.forename: Ite A
authors.initials: IA
authors.lastname: Laird-Offringa
authors.forename: Brian R
authors.initials: BR
authors.lastname: McNaughton
mesh_terms: D020121:5' Untranslated Regions; D015497:HIV-1 / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D009690:Nucleic Acid Conformation; D000071377:RNA Recognition Motif; D016601:RNA-Binding Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020349:Surface Plasmon Resonance; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics*; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D020121:5' Untranslated Regions; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C071598:TARBP1 protein, human; D000099253:Trans-Activation Responsive RNA-Binding Protein