DifferentTrypanosoma bruceiguide RNA molecules associate with an identical complement of mitochondrial proteinsin vitro

Abstract

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.

Trypanosoma brucei gBP21

“…Despite the differences in the molecular mass estimation and also some differences in the experimental conditions, the identified characteristics of that cross-link were very similar with one obvious difference: Leegwater et al (15) reported a dependence on a long U-tail on the gRNAs whereas Köller et al (16) found cross-linking even with a tail-less gRNA substrate. There is no apparent explanation for this difference but all other features support the interpretation that this cross-link is based on the interaction of gRNAs to the same protein or the same set of proteins.…”

“…Extracts were cleared from insoluble material by centrifugation at 16,000 ϫ g for 5 min at 4°C and protein concentrations were determined by dye-binding using bovine plasma ␥-globulin as a standard (26). The purification of gBP21 was followed by monitoring the binding activity to T. brucei gRNA gA6-14 (16). Cleared extracts were precipitated with ammonium sulfate at 4°C and the protein was enriched in the 50 -60% (w/v) saturation fraction.…”

“…RNA molecules (gA6-14, gA6-48, gA6-14/U, gND7-506, RNA1) were synthesized by run-off transcription from linearized plasmid DNA templates (16,32) using T7 polymerase following standard procedures. Radioactive labeling of the RNAs was achieved by either using [␣-32 P]UTP or [␣-32 P]ATP in the transcription reaction.…”

“…This was interpreted as a form of sequence specific binding to the post-transcriptionally added U-tail of gRNAs. In Trypanosoma brucei, several proteins with apparent molecular masses ranging from 124 to 9 kDa were found to cross-link to gRNA molecules (15)(16)(17)(18). The binding of the various proteins could be blocked by increasing the monovalent cation concentration and only three proteins with apparent molecular masses of 90, 21, and 9 kDa were stable at Ն100 mM potassium chloride.…”

“…All the aforementioned gRNA cross-linking studies in T. brucei identified a prominent cross-linking product in the range of 21-26 kDa, appearing as a broad radioactive signal in SDS containing polyacrylamide gels (15)(16)(17)(18). Despite the differences in the molecular mass estimation and also some differences in the experimental conditions, the identified characteristics of that cross-link were very similar with one obvious difference: Leegwater et al (15) reported a dependence on a long U-tail on the gRNAs whereas Köller et al (16) found cross-linking even with a tail-less gRNA substrate.…”