RNA-binding properties of the mitochondrial Y-box protein RBP16

• Published in: Nucleic acids research 2000-03, Vol.28 (5), p.1266-1275
• Main Authors: Pelletier, M, Miller, M.M, Read, L.K
• Format: Article
• Language: English
• Subjects: amino acid sequences; Animals; binding proteins; Binding Sites; guide RNA; messenger RNA; mitochondria; nucleotide sequences; Oligonucleotides; Protein Binding; Protozoan Proteins; RBP16 protein; RNA; RNA - metabolism; rna binding motifs; RNA-Binding Proteins - analysis; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Trypanosoma; Trypanosoma brucei; Trypanosoma brucei brucei; Y-box protein
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/28.5.1266

We have previously identified a mitochondrial Y-box protein in Trypanosoma brucei that we designated RBP16. The predicted RBP16 amino acid sequence revealed the presence of a cold-shock domain at its N-terminus and a glycine- and arginine-rich C-terminus reminiscent of an RGG RNA-binding motif. Since RBP16 is capable of interacting with different guide RNAs (gRNAs) in vitro and in vivo primarily via the oligo(U) tail, as well as with ribosomal RNAs, possible functions of RBP16 may be in kinetoplastid RNA editing and/or translation. Herein, we report experiments that further define the RNA-binding properties of RBP16. RBP16 forms a single stable complex with the gRNA gA6[14] at low protein concentration, while at higher protein concentration two stable complexes that possibly represent two different conformations are observed. Both complexes are stable at relatively high salt and moderate heparin concentrations indicating that the binding of RBP16 to gA6[14] does not rely primarily on ionic interactions. Phenylglyoxal treatment of the protein indicates that arginine residues are important in RNA binding. The minimal length of RNA sequence necessary for the binding of RBP16 was assessed by gel retardation and UV cross-linking competition assays using oligo(U) ribonucleotides of varying lengths (4-40 nt). Although RBP16 can bind to oligonucleotides as small as U(4), its affinity increases with the length of the oligo(U) ribonucleotide, with a dramatic increase in binding efficiency observed when the length is increased to 10 nt. Gel retardation assays employing T.brucei mRNAs demonstrated that, although it acts as a major binding determinant, a 3' U tail is not an absolute requirement for efficient RBP16-RNA binding. Experiments with oligonucleotides containing U stretches embedded at different positions in oligo(dC) indicated that high-affinity binding requires both a uridine stretch, as well as 5' and 3' non-specific sequences. These results suggest a model for the molecular interactions involved in RBP16-RNA binding.