Identification by UV cross-linking of oligo(U)-binding proteins in mitochondria of the insect trypanosomatid Crithidia fasciculata.
RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3' oligo(U) extension. Here we describe the identification by UV cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity. A 65-kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3' terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23-29 nucleotides. It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to mitochondrial ribosomal RNAs and to oligo(U). These proteins, however, require longer oligo(U) for binding (> 39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C). For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei. In this organism, gRNA-binding proteins of 83 kDa and 64 kDa were found with the same preference for 3'-terminal oligomeric U stretches as the C. fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein resembled that of the C. fasciculata 88-kDa and 30-kDa proteins. The possible involvement of the proteins in the editing process is discussed.
Leegwater
P
P
E. C. Slater Institute, University of Amsterdam, Academic Medical Centre, The Netherlands.
Speijer
D
D
Benne
R
R
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D016831:Crithidia fasciculata / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D007313:Insecta / Q000469:parasitology; D008928:Mitochondria / Q000378:metabolism; D009843:Oligoribonucleotides / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D017393:RNA Editing; D017394:RNA, Guide, Kinetoplastida / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D014500:Uracil Nucleotides / Q000378:metabolism*
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D014500:Uracil Nucleotides; C027916:oligo(U); D017394:RNA, Guide, Kinetoplastida