title: Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins.
abstract: The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Zubovic
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Pavelitz
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Godin
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Walker
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Suxin
authors.initials: S
authors.lastname: Zheng
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Macchi
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Varani
mesh_terms: D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering; D000071377:RNA Recognition Motif; D016601:RNA-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D016601:RNA-Binding Proteins