The stem-loop binding protein forms a highly stable and specific complex with the 3' stem-loop of histone mRNAs.
Replication-dependent histone mRNAs end in a highly conserved 26-nt stem-loop structure. The stem-loop binding protein (SLBP), an evolutionarily conserved protein with no known homologs, interacts with the stem-loop in both the nucleus and cytoplasm and mediates nuclear-cytoplasmic transport as well as 3'-end processing of the pre-mRNA by the U7 snRNP. Here, we examined the affinity and specificity of the SLBP-RNA interaction. Nitrocellulose filter-binding experiments showed that the apparent equilibrium dissociation constant (Kd) between purified SLBP and the stem-loop RNA is 1.5 nM. Binding studies with a series of stem-loop variants demonstrated that conserved residues in the stem and loop, as well as the 5' and 3' flanking regions, are required for efficient protein recognition. Deletion analysis showed that 3 nt 5' of the stem and 1 nt 3' of the stem contribute to the binding energy. These data reveal that the high affinity complex between SLBP and the RNA involves sequence-specific contacts to the loop and the top of the stem, as well the base of the stem and its immediate flanking sequences. Together, these results suggest a novel mode of protein-RNA recognition that forms the core of a ribonucleoprotein complex central to the regulation of histone gene expression.
Battle
D J
DJ
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA
Doudna
J A
JA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D002460:Cell Line; D016384:Consensus Sequence; D006657:Histones / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D018411:Spodoptera; D013816:Thermodynamics; D014162:Transfection; D014981:Xenopus; D029867:Xenopus Proteins; D039221:mRNA Cleavage and Polyadenylation Factors
D006657:Histones; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C092663:SLBP protein, human; C490491:SLBP1 protein, Xenopus; D029867:Xenopus Proteins; D039221:mRNA Cleavage and Polyadenylation Factors