Different Trypanosoma brucei guide RNA molecules associate with an identical complement of mitochondrial proteins in vitro.
RNA editing is a mitochondrial transcript maturation process which evolved in kinetoplastid protozoa. It entails the insertion and deletion of exclusively uridine nucleotides directed by gRNAs into pre-mRNAs. Other participating components are not currently known. The aim of this study was to identify mitochondrial proteins that are in direct physical contact with gRNAs thereby possibly involved in the editing reaction. At low monovalent cation concentration (30 mM KCl) 8 polypeptides with apparent molecular weights ranging from 124 to 9 kDa specifically cross-linked to gRNAs. Three of the proteins, 90, 21, and 9 kDa in size, were able to bind at higher salt concentrations (> or = 100 mM) indicating an enhanced affinity to the gRNA molecules. No cross-links were identified at > or = 250 mM KCl. Four gRNAs, specific for different editing domains of the ATPase 6 and ND7 pre-mRNAs, were in contact with the same set of mitochondrial polypeptides suggesting the assembly of an identical RNP complex that does not include pre-mRNA molecules. The binding of the 90 kDa protein was sensitive to the presence of U-nucleotides at the 3'-end of the gRNAs and could specifically be blocked by modifying free sulfhydryl groups. The interaction with the 124 kDa polypeptide was inhibited by vanadyl ribonucleosides, implicating a role for 2', 3' hydroxyl groups in the gRNA-protein interaction.
Laboratorium für Molekulare Biologie, Genzentrum, Martinsried, Germany.
J
J
Köller
G
G
Nörskau
A S
AS
Paul
K
K
Stuart
H U
HU
Göringer
D000818:Animals; D008928:Mitochondria / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism; D017393:RNA Editing; D017394:RNA, Guide, Kinetoplastida / Q000378:metabolism*; D016053:RNA, Protozoan / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D015800:Protozoan Proteins; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; D017394:RNA, Guide, Kinetoplastida