title: Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.
abstract: Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.
authors: Daniel T
authors: DT
authors: Peters
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.|Department of Biology, University of York , York YO10 5DD, United Kingdom.
authors: Herman K H
authors: HK
authors: Fung
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.
authors: Vladimir M
authors: VM
authors: Levdikov
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.
authors: Tobias
authors: T
authors: Irmscher
authors: Department of Biology, University of York , York YO10 5DD, United Kingdom.
authors: Fiona C
authors: FC
authors: Warrander
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.
authors: Sandra J
authors: SJ
authors: Greive
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.
authors: Oleg
authors: O
authors: Kovalevskiy
authors: Department of Biology, University of York , York YO10 5DD, United Kingdom.
authors: Harry V
authors: HV
authors: Isaacs
authors: Department of Biology, University of York , York YO10 5DD, United Kingdom.
authors: Mark
authors: M
authors: Coles
authors: York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.
authors: Alfred A
authors: AA
authors: Antson
mesh_terms: D005454:Fluorescence Polarization; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: C000715187:Lin28A protein, human; C574861:MIRN363 microRNA, human; D035683:MicroRNAs; D016601:RNA-Binding Proteins