title: KH domains with impaired nucleic acid binding as a tool for functional analysis.
abstract: In eukaryotes, RNA-binding proteins that contain multiple K homology (KH) domains play a key role in coordinating the different steps of RNA synthesis, metabolism and localization. Understanding how the different KH modules participate in the recognition of the RNA targets is necessary to dissect the way these proteins operate. We have designed a KH mutant with impaired RNA-binding capability for general use in exploring the role of individual KH domains in the combinatorial functional recognition of RNA targets. A double mutation in the hallmark GxxG loop (GxxG-to-GDDG) impairs nucleic acid binding without compromising the stability of the domain. We analysed the impact of the GDDG mutations in individual KH domains on the functional properties of KSRP as a prototype of multiple KH domain-containing proteins. We show how the GDDG mutant can be used to directly link biophysical information on the sequence specificity of the different KH domains of KSRP and their role in mRNA recognition and decay. This work defines a general molecular biology tool for the investigation of the function of individual KH domains in nucleic acid binding proteins.
authors.affiliation: Molecular Structure Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: David
authors.initials: D
authors.lastname: Hollingworth
authors.forename: Adela M
authors.initials: AM
authors.lastname: Candel
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Nicastro
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Martin
authors.forename: Paola
authors.initials: P
authors.lastname: Briata
authors.forename: Roberto
authors.initials: R
authors.lastname: Gherzi
authors.forename: Andres
authors.initials: A
authors.lastname: Ramos
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D009154:Mutation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism
chemical_list: C105789:KHSRP protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D012313:RNA