Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):13944-9. doi: 10.1073/pnas.0604476103. Epub 2006 Sep 8.

Abstract

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Eosinophil Cationic Protein / metabolism
  • Gene Expression Regulation
  • Leishmania / genetics
  • Leishmania / metabolism
  • Mitochondria / genetics
  • Mitochondria / metabolism
  • Molecular Sequence Data
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • RNA / genetics
  • RNA / metabolism*
  • RNA Editing*
  • RNA Interference
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Trypanosoma brucei brucei / genetics
  • Trypanosoma brucei brucei / metabolism
  • Uridine / metabolism*

Substances

  • Protozoan Proteins
  • RNA-Binding Proteins
  • Recombinant Proteins
  • RNA
  • Eosinophil Cationic Protein
  • Uridine