Identification by UV Cross-Linking of oligo(U)-binding Proteins in Mitochondria of the Insect Trypanosomatid Crithidia Fasciculata

Eur J Biochem. 1995 Feb 1;227(3):780-6. doi: 10.1111/j.1432-1033.1995.tb20201.x.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding, Competitive
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Carrier Proteins / radiation effects
  • Crithidia fasciculata / genetics
  • Crithidia fasciculata / metabolism*
  • Cross-Linking Reagents
  • Insecta / parasitology
  • Mitochondria / metabolism
  • Oligoribonucleotides / metabolism*
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Protozoan Proteins / radiation effects
  • RNA Editing
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism
  • Species Specificity
  • Trypanosoma brucei brucei / genetics
  • Trypanosoma brucei brucei / metabolism
  • Ultraviolet Rays
  • Uracil Nucleotides / metabolism*

Substances

  • Carrier Proteins
  • Cross-Linking Reagents
  • Oligoribonucleotides
  • Protozoan Proteins
  • RNA, Guide
  • RNA, Protozoan
  • Uracil Nucleotides
  • oligo(U)