Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains.
Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain. To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with Kd values of 12-140 nM. The highest affinity RNA sequences (Kd approximately 12-40 nM) contained a UAAA motif; mutation to UACA abolished binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. Deletion of the KH domain or mutation of KH domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1 abolished G8-5 binding. Our results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.
Lin
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Taylor
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SJ
Shalloway
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D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D002238:Carbohydrate Epimerases; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D019976:Cloning, Organism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005600:Fragile X Syndrome / Q000235:genetics; D016147:Genes, Tumor Suppressor; D015801:Helminth Proteins / Q000378:metabolism; D006801:Humans; D007658:Ketone Oxidoreductases; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; C074561:GAP-associated protein p62; C094448:GLD-1 protein, C elegans; D015801:Helminth Proteins; C489559:KHDRBS1 protein, human; C513253:Khdrbs1 protein, mouse; D009843:Oligoribonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; C028277:TSTA3 protein, human; D007658:Ketone Oxidoreductases; D002238:Carbohydrate Epimerases