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

• Published in: Nucleic acids research 2001-07, Vol.29 (14), p.2950-2962
• Main Authors: Blom, D, Burg Jv, Breek, C K, Speijer, D, Muijsers, A O, Benne, R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cloning, Molecular; Crithidia fasciculata; Crithidia fasciculata - genetics; DNA, Complementary - chemistry; DNA, Complementary - genetics; gBP27 protein; gBP29 protein; gRNA-binding protein; guide RNA; guide RNA-binding protein; Molecular Sequence Data; Precipitin Tests; Protein Binding; Protozoan Proteins; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA - genetics; RNA, Guide - metabolism; RNA, Messenger - metabolism; RNA, Mitochondrial; RNA, Protozoan - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Trypanosoma brucei brucei - genetics
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/29.14.2950

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning 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 approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. 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 proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs 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-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.