Biomedical subjects
Publications and source records attributed to S Baskerville.
The three-dimensional structures of RNAs are notoriously difficult to determine. Functional comparisons of variant molecules and cross-linking experiments are providing new information for structural modeling.
In vitro selection, or SELEX, has been used both to characterize the interaction of natural nucleic acids with proteins and to generate novel nucleic acid-binding species, or aptamers. Although numerous reports have demonstrated the power of the technique, they have not expanded on the methodologies that can be used for selection. This review focuses on the considerations and problems involved in selecting protein-binding aptamers from a random-sequence RNA pool. As an illustration, we describe two approaches to selecting aptamers to a particular target, the HTLV-I Rex protein. In the first, complete randomization is used to find an artificial, high-affinity RNA binding site. In the second, the contributions of individual nucleotides and/or base pairs to the natural Rex-binding element are determined by mutating the wild-type sequence and selecting active binding variants.
Interactions between the Rex protein of HTLV-1 and the genomic Rex-binding element (XBE) mediate the cytoplasmic transport of viral mRNAs. However, it is uncertain which RNA sequences and structures contribute to Rex recognition. A portion of the viral genome that spanned the XBE was partially randomized, and functional Rex-binding variants were selected. Alignment of selected Rex-binding sequences revealed positions that were functionally conserved between different molecules. A model is presented in which a subset of the selected residues are in direct contact with Rex. Positions that covaried with one another were also found. These covariations support a secondary-structural model in which a central paired stem is symmetrically flanked by two bulge loops. On the basis of this model, site-directed mutations of the XBE were constructed and each half molecule was found to bind independently to Rex. The functional residues and secondary structures in the XBE half molecules bear a remarkable resemblance to the transactivation response region element of HIV-1. Since the transactivation response region element is known to interact specifically with arginine residues in the Tat protein, these results suggest that the XBE binds to the arginine-rich RNA-binding domain of Rex in a similar manner. This model is supported by the selection data.
The Tat and Rev proteins of HIV-1 and the Rex protein of HTLV-I do not interact with their cognate ligands via a particular structural motif but instead specifically recognize RNA molecules by using agglomerations of arginine residues (1). These proteins are members of the so-called arginine-rich motif (ARM) family. There is little data to support (or contradict) the hypothesis that a few simple arginine:RNA interactions govern how ARMs recognize their viral targets. Not only is it unclear how ARM proteins other than Tat interact with their cognate RNA ligands, for the most part it is not even known how structurally complex these RNA ligands are. In order to fully explore the range of RNA sequences and structures that can bind to ARMs we have carried out in vitro genetic selections with two disparate viral proteins: Rev and Rex.