The stem-loop binding protein forms a highly stable and specific complex with the 3' stem-loop of histone mRNAs

RNA. 2001 Jan;7(1):123-32. doi: 10.1017/s1355838201001820.

Abstract

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.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Calorimetry
  • Cell Line
  • Consensus Sequence
  • Histones / metabolism
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Nucleic Acid Conformation*
  • RNA, Messenger / chemistry*
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Ribosomes / metabolism
  • Spodoptera
  • Thermodynamics
  • Transfection
  • Xenopus
  • Xenopus Proteins*
  • mRNA Cleavage and Polyadenylation Factors*

Substances

  • Histones
  • Nuclear Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Proteins
  • SLBP protein, human
  • SLBP1 protein, Xenopus
  • Xenopus Proteins
  • mRNA Cleavage and Polyadenylation Factors