OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity.
KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3' oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem-loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.
Kala
Smriti
S
Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Quebec, Canada.
Salavati
Reza
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism
C510680:KREPA4 protein, Trypanosoma brucei; D015800:Protozoan Proteins; D012322:RNA Precursors; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017394:RNA, Guide, Kinetoplastida