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-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.
Blom
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Department of Biochemistry, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Burg Jv
Breek
C K
CK
Speijer
D
D
Muijsers
A O
AO
Benne
R
R
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016831:Crithidia fasciculata / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D015800:Protozoan Proteins; D012313:RNA / Q000235:genetics*; D017394:RNA, Guide, Kinetoplastida / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics
D018076:DNA, Complementary; D015800:Protozoan Proteins; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C104816:gBP21 protein, Trypanosoma brucei; C433832:gBP27 protein, Crithidia fasciculata; C433833:gBP29 protein, Crithidia fasciculata; D012313:RNA; D017394:RNA, Guide, Kinetoplastida