Optimized RNA targets of two closely related triple KH domain proteins, heterogeneous nuclear ribonucleoprotein K and alphaCP-2KL, suggest Distinct modes of RNA recognition

• Published in: The Journal of biological chemistry 2001-05, Vol.276 (20), p.17484
• Main Authors: Thisted, T, Lyakhov, D L, Liebhaber, S A
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Binding Sites; Consensus Sequence; DNA Primers - chemistry; DNA-Binding Proteins; Heterogeneous-Nuclear Ribonucleoprotein K; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Mice; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemistry; Poly C - metabolism; Polymerase Chain Reaction; Recombinant Proteins - metabolism; Ribonucleases; Ribonucleoproteins - metabolism; RNA - chemistry; RNA - metabolism; RNA Processing, Post-Transcriptional; RNA-Binding Proteins - metabolism; Substrate Specificity; Transcription Factors
• ISSN: 0021-9258

The KH domain mediates RNA binding in a wide range of proteins. Here we investigate the RNA-binding properties of two abundant RNA-binding proteins, alphaCP-2KL and heterogeneous nuclear ribonucleoprotein (hnRNP) K. These proteins constitute the major poly(C) binding activity in mammalian cells, are closely related on the basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of post-transcriptional controls. By using SELEX, we have obtained sets of high affinity RNA targets for both proteins. The primary and secondary structures necessary for optimal protein binding were inferred in each case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for alphaCP-2KL and hnRNP K were both enriched for cytosine bases and were presented in a single-stranded conformation. In contrast to these shared characteristics, the optimal target sequence for hnRNP K is composed of a single short "C-patch" compatible with recognition by a single KH domain whereas that for alphaCP-2KL encompassed three such C-patches suggesting more extensive interactions. The binding specificities of the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized alphaCP-2KL-binding site was supported by comparison of SELEX target structure with that of the native human alpha-globin 3'-untranslated region. These data indicate that modes of macromolecular interactions of arrayed KH domains can differ even among closely related KH proteins and that binding affinities are substantially dependent on the presentation of the target site within the RNA secondary structure.