ORIGINS OF BINDING-SPECIFICITY OF THE A1 HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN

The A1 heterogeneous nuclear ribonucleoprotein (hnRNP) is the best stu died of the ''core'' hnRNP proteins that are tightly associated with h eterogeneous nuclear RNA (hnRNA) within eukaryotic nuclei. Previous st udies suggested that hnRNP A1 preferentially binds (under nonequilibri um conditions) to the pyrimidine-rich span of sequence at the 3'-splic e site of most introns [Swanson, M.S., & Dreyfuss, G. (1988) EMBO J. 1 1, 3519-3529; Buvoli et al. (1990) Nucleic Acids Res. 18, 6595-6600; I shikawa et al. (1993) Mol. Cell. Biol. 13, 4301-4310]. Recently, Burd and Dreyfuss [(1994) EMBO J. 13, 1197-1204] used selection/amplificati on from pools of random sequence RNA to uncover an even higher-affinit y A1 oligo that contained two copies of a high-affinity consensus sequ ence, UAGGGU/A. We have extended these studies by using a fluorescence assay to characterize the equilibrium binding properties of A1 to eac h of these oligonucleotides. By also characterizing the binding of A1 to sequence-randomized control oligonucleotides, we have been able to better evaluate the inherent ''sequence-specific'' binding properties of A1. Although these studies indicate that under equilibrium conditio ns A1 cannot specifically recognize the beta-globin, 3'-splice site DN A oligo analogue studied by Buvoli et al. (1990), they confirmed the h igh-affinity binding to the ''winner'' 20-mer RNA that was uncovered v ia selection/amplification and that has the sequence UAUGAUAGGGACUUAGG GUG (Burd & Dreyfuss, 1994). In 0.1 M NaCl, we found that A1 has simil ar to 100-fold higher affinity for this winner sequence than it does f or either a randomized version of this sequence or a 20-mer oligo corr esponding to an unrelated beta-globin intron sequence, This winner RNA oligo aggregates in solution to form an apparent dimer that may repre sent a G-quartet resulting from dimerization of two Hoogsteen base-pai red hairpins. On the basis of salt sensitivity studies carried out wit h various fragments of A1, the ability of A1 to discriminate the winne r sequence from its randomized control results primarily from increase d ionic interactions with the glycine-rich, COOH terminal domain of A1 that extends from residue 196 to 319. Nonetheless, most of the overal l energy of binding for the A1 winner complex results from determinant s that are resident within the first 195 residues of A1. The unique ab ility of the winner sequence (but not its sequence-randomized control) to form a higher-order aggregate, which may correspond to a G-tetrad, appears to facilitate the additional ionic interactions with the COOH terminal domain. Taken together, these data suggest the need to reeva luate possible and probable functions of A1 in vivo.