In vitro genetic analysis of the RNA binding site of vigilin, a multi- KH-domain protein

The steps between gene transcription and mRNA translation, which include nuclear RNA processing, mRNA trafficking, and cytoplasmic mRNA degradation, are increasingly seen as important regulatory sites in diverse cellular processes (5, 6, 52). Many of these steps in mRNA metabolism appear to be regulated by RNA binding proteins containing K-homology (KH) domains (14). While a detailed picture of KH-domain–RNA interaction is not yet available, in several cases proteins containing KH domains have been shown to bind RNA (13, 14, 21, 57). Some KH-domain proteins are clinically significant, including FMR1 protein (57), which is involved in fragile X syndrome, the major cause of heritable human mental retardation, and Nova-1, which is important in the motor control disorder paraneoplastic opsoclonus-ataxia (12, 13). KH-domain proteins which affect nuclear RNA splicing include Mer1, SF1, and PSI (1, 46, 55), while the α-poly(C) binding protein plays a role in the cytoplasmic stability of globin mRNA (68). Prokaryotic KH-domain proteins are highly diverse and include NusA, polyribonucleotide:orthophosphate nucleotidyltransferase, CsrA, and ribosomal protein S3 (27, 40, 58). Since the RNA binding sites and mechanisms of action of many KH-domain proteins remain obscure, the question of whether KH-domain proteins can preferentially recognize and bind to specific RNA binding sites remained unresolved. In an important recent paper, Buckanovich and Darnell used in vitro genetic selection with purified recombinant Nova-1 to identify RNAs which bind Nova-1 with high affinity. Nova-1 and inhibitory glycine receptor α2 RNAs were then shown to contain consensus binding sites which bind to Nova-1 through a KH domain (12). One important but little-understood KH-domain protein is vigilin. Vigilin initially was cloned from chicken chondrocytes and then was shown to be up-regulated in rapidly dividing cells and to be present in all vertebrate cell lines tested, in nematodes, and perhaps in yeast (35, 47, 49, 50, 54, 69). With its 14 KH domains, vigilin has been used as a model protein for the KH domains in FMR1 and other proteins in a seminal study solving the structure of a vigilin KH domain uncomplexed to RNA (45). However, the intracellular RNA targets of this ubiquitous, model protein and its functions remained elusive. We had shown that estrogen specifically stabilizes the hepatic mRNA coding for the egg yolk precursor protein vitellogenin, increasing its half life from 16 to 500 h (10). The estrogen-mediated stabilization of vitellogenin mRNA requires estrogen receptor (48) and association of the mRNA with polysomes (9) and involves the 3′ untranslated region (UTR) of the mRNA (48). We identified an estrogen-inducible protein which binds specifically to a segment of the vitellogenin mRNA 3′ UTR (25). We recently unambiguously identified this protein as Xenopus vigilin (26). Our observations that the protein we now refer to as vigilin is found in several human cell lines and in several Xenopus cell lines and tissues and that testosterone induces vigilin in Xenopus muscle suggested a wider role for vigilin in eukaryotic mRNA metabolism (24). Despite the wide distribution of vigilin, binding sites in mRNAs other than vitellogenin mRNA had not been identified. To allow prediction of vigilin binding sites in human mRNAs, we first defined the vigilin binding site by using iterative in vitro genetic selection. In vitro genetic selection or systematic evolution of ligands by exponential enrichment has been used to identify RNA (and DNA) ligands which interact with proteins or small molecules (28). Although an early study (8) and several subsequent studies used DNA gel mobility shifts to separate free and protein-bound DNA, in vitro genetic selection with an RNA gel mobility shift has not been described. RNA binding sites have been identified for several proteins by using in vitro genetic analysis (11, 12, 15, 31, 39, 56, 62, 63, 66). Those studies used purified RNA binding proteins. For example, in early studies, in vitro selection of RNA binding sites was used to identify the structure of the human immunodeficiency virus (HIV) Rev binding site (3) and the binding preferences of splicing factors (31, 56, 62), hnRNPs (15, 29), and the autoimmune RNA binding protein Hel-N1 (39). Using in vitro genetic selection of gel-shifted RNA-protein complexes, we have extended this technique to proteins present in relatively crude extracts. Since this work was carried out in parallel with studies aimed at the identification of the vitellogenin mRNA 3′ UTR binding protein, we had to employ a relatively crude polysome extract. We first selected RNA binding sites with an increased affinity for vigilin. We then used an RNA selected for increased binding to vigilin to carry out a novel in vitro selection for random point mutations that markedly reduced binding of vigilin. Together with deletion analysis and RNA footprinting, the in vitro genetic analysis enabled us to identify structural and sequence elements important for the interaction of vigilin with RNA. Using this information, we scanned the human sequence database and predicted and subsequently identified a strong vigilin binding site near the 3′ end of human dystrophin mRNA. We then analyzed binding of purified recombinant human vigilin to the selected up- and down-binding RNAs and demonstrated that vigilin exhibits strong preferences for binding to specific RNAs.