title: Both RNA-binding domains in heterogenous nuclear ribonucleoprotein A1 contribute toward single-stranded-RNA binding.
abstract: Heterogenous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A1 are tightly associated with heterogenous nuclear RNAs (hnRNAs) within eukaryotic nuclei and are thought to be involved in hnRNA processing and splice site selection. The NH2-terminal two-thirds of hnRNP A1 contains two 92-amino acid RNA binding domains (RBDs) that are arranged in tandem and are more than 30% homologous with each other. Following this region is a flexible glycine-rich COOH-terminal domain. We have studied the nucleic acid binding properties of the two isolated RBDs (residues 1-92 and 93-184, respectively) and of A1 fragments corresponding to residues 1-184 and 1-196 (i.e., the latter fragment is called UP1) in order to evaluate their relative contributions to A1 binding. We have determined that the individual RBDs of A1 bind poly[r(epsilon A)], a fluorescent single-stranded RNA (ssRNA), with a surprisingly low apparent association constant of only 1.5 x 10(4) M-1 (1-92) and 4.5 x 10(4) M-1 (93-184), respectively. We hypothesize that this low affinity represents a basal level of binding that is common to most RBD-containing proteins. Oligonucleotide binding studies suggest the interaction site size for the 93-184 fragment is approximately 4 nucleotides or less and salt sensitivity studies indicate that only about 27% of the free energy of binding of this RBD derives from ionic interactions. Since the affinity of the 1-184 fragment is at least 10-fold above that of either of its component RBDs, both must contribute to binding. This conclusion is further supported by the increased occluded site size of 1-184 (n = 14 +/- 2), as compared to its 93-184 RBD (n = 6 +/- 1), and by the biphasic binding that was observed for the UP1:poly(U) interaction at pH 6.0. Our finding that the affinity of the 1-184 fragment is 1000-fold less than the product of the affinities of its 1-92 and 93-184 RBDs is consistent with these domains being joined by a flexible linker. By comparing the affinities of the 1-184 fragment with that for A1, we conclude that together the two RBDs in A1 account for only 53% of the free energy of A1 binding. Comparative binding studies with UP1 demonstrate that the short region spanning residues 185-->195 represents an important determinant of the binding affinity of A1 and, since this region contains a site of dimethylation, it may provide a mechanism for regulating the affinity of A1 for specific nucleic acid targets.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510-8024.
authors.forename: Y
authors.initials: Y
authors.lastname: Shamoo
authors.forename: N
authors.initials: N
authors.lastname: Abdul-Manan
authors.forename: A M
authors.initials: AM
authors.lastname: Patten
authors.forename: J K
authors.initials: JK
authors.lastname: Crawford
authors.forename: M C
authors.initials: MC
authors.lastname: Pellegrini
authors.forename: K R
authors.initials: KR
authors.lastname: Williams
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004563:Electrochemistry; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride; D013816:Thermodynamics
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009841:Oligonucleotides; D010446:Peptide Fragments; D012261:Ribonucleoproteins; D011072:Poly U; D012965:Sodium Chloride; D012313:RNA; D004247:DNA