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RNA-binding properties of the mitochondrial Y-box protein RBP16
M Pelletier1, M M Miller, L K Read
1Department of Microbiology and Center for Microbial Pathogenesis, SUNY Buffalo School of Medicine, 138 Farber Hall, Buffalo, NY 14214, USA.
Abstract:
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.
Insights
Mitochondrial protein RBP16 in Trypanosoma brucei binds guide RNAs (gRNAs) and ribosomal RNAs. Arginine residues and oligo(U) sequences are key for RBP16-RNA interactions, suggesting roles in RNA editing and translation.
Area of Science:
- Molecular Biology
- Parasitology
- Biochemistry
Background:
- A mitochondrial Y-box protein, RBP16, was identified in Trypanosoma brucei.
- RBP16 possesses a cold-shock domain and an RGG RNA-binding motif.
- RBP16 interacts with guide RNAs (gRNAs) and ribosomal RNAs, suggesting roles in RNA editing and translation.
Purpose of the Study:
- To further characterize the RNA-binding properties of RBP16.
- To elucidate the molecular mechanisms underlying RBP16-RNA interactions.
Main Methods:
- Gel retardation assays
- UV cross-linking competition assays
- Phenylglyoxal treatment
Main Results:
- RBP16 forms stable complexes with gRNA gA6[14] that are salt- and heparin-resistant.
- Arginine residues are crucial for RBP16's RNA binding.
- High-affinity binding requires oligo(U) stretches and flanking non-specific sequences, with affinity increasing up to 10 nt.
- A 3' U tail is not essential for efficient RBP16-RNA binding to T. brucei mRNAs.
Conclusions:
- RBP16 exhibits specific RNA-binding characteristics involving uridine-rich sequences and arginine residues.
- These findings provide insights into the molecular interactions of RBP16, supporting its potential roles in kinetoplastid RNA processing and translation.