title: Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.
abstract: Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Shuyun
authors.initials: S
authors.lastname: Dong
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Caleb
authors.initials: C
authors.lastname: Cassidy-Amstutz
authors.forename: Gang
authors.initials: G
authors.lastname: Lu
authors.forename: Rebecca
authors.initials: R
authors.lastname: Bigler
authors.forename: Mark R
authors.initials: MR
authors.lastname: Jezyk
authors.forename: Chunhua
authors.initials: C
authors.lastname: Li
authors.forename: Traci M Tanaka
authors.initials: TM
authors.lastname: Hall
authors.forename: Zefeng
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; C525831:Nop9 protein, S cerevisiae; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C075059:fem-3 protein, C elegans; D012313:RNA; D003596:Cytosine