RNA-protein interactions in the ribonucleoprotein T-complexes in a mitochondrial extract from Leishmania tarentolae

Abstract

We have investigated protein-RNA interactions and the incorporation of [α- 32P]UTP into the guide RNA and mRNA components of the ‘T-complexes’ in a mitochondrial extract from Leishmania tarentolae. The terminal uridylyl transferase-containing complex T-IV is probably involved in the maturation of the 3′-oligo(U) tail of the gRNAs, but the biological function and biochemical nature of the remaining T-complexes is not known. We have found that the relative extent of labeling of the RNA components is dependent on the UTP concentration: at low levels, the main endogenous RNA components labeled are the gRNAs in T-IV; at higher levels, the mRNAs in all of the T-complexes are preferentially labeled. We also show a tentative correlation in the migration pattern of UTP-labeled T-complexes and complexes which bind exogenous labeled RNA. The relative extent of binding to specific complexes is dependent upon the type of RNA. Most of the interactions between the labeled RNAs and proteins can be disrupted by heparin or a large excess of rRNA, but two labeled complexes were resistant to competition. Most of the binding of labeled exogenous gRNA is disrupted by competition with a large excess of rRNA, but predigestion of the extract with micrococcal nuclease and saturation with rRNA uncovered a high affinity complex, which involves at least two proteins interacting with the bound gRNAs. A knowledge of the RNA and protein components may aid in understanding the biological roles of these RNP complexes.