Cloning and characterization of two guide RNA-binding proteins from mitochondria of Crithidia fasciculata: gBP27, a novel protein, and gBP29, the orthologue of Trypanosoma brucei gBP21

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptiona lly edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the edit ing process envisage a series of consecutive 'cut-and-paste' reactions, car ried out by a complex RNP machinery. Here we report on the purification, cl oning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of similar to 10.0. gBP27 has no homology to known proteins , but gBP29 is the C. fasciculata orthologue of gBP21 from Trypanosoma bruc ei, a gRNA-binding protein that associates with active RNA editing complexe s. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these pr oteins. Immunoprecipitates; of mt extracts generated with antibodies agains t gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNA s and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-elu ted with gRNAs from gel filtration columns in the high molecular weight ran ge. Together, these results suggest that the proteins are part of a large m acromolecular complex(es). We infer that gBP29 and gBP27 are components of the C. fasciculata editing machinery that may interact with gRNAs.