Abstract
Inhibition of RNA editing by down-regulation of expression of the mitochondrial RNA editing TUTase 1 by RNA interference had profound effects on kinetoplast biogenesis in Trypanosoma brucei procyclic cells. De novo synthesis of the apocytochrome b and cytochrome oxidase subunit I proteins was no longer detectable after 3 days of RNAi. The effect on protein synthesis correlated with a decline in the levels of the assembled mitochondrial respiratory complexes III and IV, and also cyanide-sensitive oxygen uptake. The steady-state levels of nuclear-encoded subunits of complexes III and IV were also significantly decreased. Because the levels of the corresponding mRNAs were not affected, the observed effect was likely due to an increased turnover of these imported mitochondrial proteins. This induced protein degradation was selective for components of complexes III and IV, because little effect was observed on components of the F(1).F(0)-ATPase complex and on several other mitochondrial proteins.
Highlights
Gene expression in the kinetoplast-mitochondrion of trypanosomatid protists involves a unique post-transcriptional mRNA maturation process, termed RNA editing, whereby Uresidues are inserted to or deleted from a pre-edited transcript, and a translatable reading frame is created [1,2,3,4,5]. 12 of the 18 protein-coding genes in the maxicircle component of the mitochondrial or kinetoplast DNA in Trypanosoma brucei or Leishmania tarentolae encode transcripts that require varying degrees of editing for translation competence [6]
Because Cyb is translated from an edited mRNA, whereas cytochrome oxidase subunit I (COI) is translated from a never-edited mRNA, this represents a useful system to investigate the effects of RNA editing on mitochondrial translation
This is not unexpected, because the COI polypeptide from L. tarentolae was found previously to be refractory to trypsin digestion,3 and a similar property is anticipated for the T. brucei protein
Summary
Apocytochrome b; COI, -II, -III, cytochrome oxidase subunits I, II, and III; TUTase, terminal uridylyl transferase; RNAi, RNA interference; TAO, trypanosome alternative oxidase;. The information for selection of editing sites resides in small “guide” RNAs that are mainly encoded in the kinetoplast DNA minicircles [7, 14]. These RNA molecules have 3Ј oligo-U tails [15], which are added posttranscriptionally by the RET1 TUTase [16, 17]. Editing provides translatable transcripts, inhibition of editing should affect protein synthesis and, the assembly of respiratory complexes in the kinetoplast. We have inhibited RNA editing in T. brucei by down-regulation of RET1 and have investigated the effects on mitochondrial translation and stability of the mitochondrial respiratory complexes
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