ASSEMBLY OF MITOCHONDRIAL RIBONUCLEOPROTEIN COMPLEXES INVOLVES SPECIFIC GUIDE RNA (GRNA)-BINDING PROTEINS AND GRNA DOMAINS BUT DOES NOT REQUIRE PREEDITED MESSENGER-RNA

RNA editing in kinetoplastids probably employs a macromolecular comple x, the editosome, that is likely to include the guide RNAs (gRNAs) whi ch specify the edited sequence. Specific ribonucleoprotein (RNP) compl exes which form in vitro with gRNAs (H. U. Goringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several fa ctors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Pree dited mRNA promotes the in vitro formation of the four major gRNA-cont aining RNP complexes under some conditions but is required for the for mation of only a subcomponent of one complex. The 5' gRNA sequence enc ompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail a re both important in complex formation, since their deletion results i n a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in conta ct with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein b inds specifically to gRNA oligo(U) tails. The gRNA-binding proteins ex hibit a differential distribution between the four in vitro-formed com plexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRN As are used for complex formation.