Abstract
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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Amino Acid Sequence
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Animals
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Base Sequence
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Binding Sites
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Caenorhabditis elegans / metabolism
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Caenorhabditis elegans Proteins*
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Carbohydrate Epimerases*
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism
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Cloning, Organism
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / metabolism*
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Fragile X Syndrome / genetics
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Genes, Tumor Suppressor
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Helminth Proteins / metabolism
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Humans
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Ketone Oxidoreductases*
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Mice
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Oligoribonucleotides / chemistry
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Oligoribonucleotides / metabolism
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Phosphoproteins / chemistry*
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Phosphoproteins / metabolism*
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Polymerase Chain Reaction
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RNA / chemistry*
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RNA / metabolism*
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RNA-Binding Proteins / chemistry*
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RNA-Binding Proteins / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Substrate Specificity
Substances
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Adaptor Proteins, Signal Transducing
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Caenorhabditis elegans Proteins
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Carrier Proteins
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DNA-Binding Proteins
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Dok1 protein, mouse
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GAP-associated protein p62
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GLD-1 protein, C elegans
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Helminth Proteins
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KHDRBS1 protein, human
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Khdrbs1 protein, mouse
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Oligoribonucleotides
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Phosphoproteins
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RNA-Binding Proteins
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Recombinant Proteins
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RNA
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TSTA3 protein, human
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Ketone Oxidoreductases
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Carbohydrate Epimerases